NO832292L - MODIFIED LABELED NUCLEOTIDES AND POLYNUCLEOTIDES, AND THEIR PREPARATION, USE AND DIRECTION - Google Patents

Abstract

Disclosed is a protein to which a moiety capable of chelating an ion is directly or indirectly attached, said attachment being characterized in not interfering substantially with the ability of the moiety to chelate ions.

Description

It is known to produce nucleotides or polynucleotides

which is radioactively labeled, such as with isotopes of

3 32 14 125 hydrogen ( H), phosphorus (P), carbon ( C) or iodine ( I). Such radioactively labeled compounds are useful for detecting, monitoring, localizing and isolating nucleic acid and other molecules of scientific or clinical interest. However, the use of radioactively labeled materials means

time unfortunately the danger due to the radiation. And watched

due to the relatively short half-life of the radioactive materials used to label such compounds or materials, the resulting labeled compounds or materials have a correspondingly short shelf life.

It is suggested to chemically label compounds of interest,

like for example. nucleotides and polynucleotides, to overcome or avoid the dangers and difficulties associated with such compounds or materials when radiolabelled. In the article by P.R. Langer, A.A. Waldrop and D.C. Ward entitled "Enzymatic Synthesis of Biotin-

Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes",

in Proe. Nati. Acad. Sci. United States, vol. 78, No. 11, pages 6633-6637, November, 1981, analogs of dUTP and UTP are described which contain a biotin molecule bound to the C-5 position of the pyrimidine ring by means of an alkylamine linker.

The biotin-labelled nucleotides are effective substrates for

a variety of DNA and RNA polymerases in vitro. Polynucleotides containing low levels of biotin substitution (50 molecules or fewer per kilobase) have denaturation, reassociation, and hybridization properties similar to unsubstituted controls. Biotin-labeled polynucleotides, both single- and double-linked, are selectively and quantitatively retained on avidin-Sepharose, even after extensive washing with 8M urea,

6M guanidine hydrochloride or 99% formamide. In addition, biotin-labeled nucleotides can be selectively immunoprecipitated in the presence of antibiotin antibodies and Staphylococcus aurea, Protein A. These unique properties of biotin-labeled polynucleo-

times make them useful affinity probes for the detection and isolation of particular DNA and RNA sequences. It is stated in the article that the content of the article is covered by a pending U.S. patent application.

What is described in this article and in the above-mentioned patent application is included here and made part of this description.

The patent application referred to in the above article is U.S. Pat. patent application no. 255,223 filed on 17 April 1981. What is described in this patent application no. 255,223 is incorporated herein and made part of this description. In the above-mentioned U.S. application is the subject of the above article described and in addition it is described that connections with the structure:

wherein B represents a purine, deazapurine, or pyrimidine moiety covalently attached to the C 1' position of the sugar moiety, provided that when B is purine or 7-deazapurine, it is attached to the N 9 position of the purine or deazapurine , and when B is pyrimidine, it is attached with the N - position,

where A represents a moiety consisting of at least three carbon atoms capable of forming a detectable complex with a polypeptide when the compound is introduced into a double-linked ribonucleic acid, deoxyribonucleic acid duplex or a DNA-RNA hybrid,

where the dotted line represents a chemical bond connecting B and A, provided that if B is purine the bond is attached to the 8-position of the purine, if B

is 7-deazapurine, the bond is attached to the 7-position of the deazapurine, and if B is pyrimidine, the bond is attached to the 5-position of the pyrimidine, and

in which each of x, y, and z represents

widely used as probes in biomedical research and recombinant DNA technology.

Particularly useful are compounds which are encompassed by this structure and which additionally have one or more of the following characteristics: A is non-aromatic, A is at least C,.,

the chemical bond joining B and A comprises an α-olefinic bond, Å is biotin or iminobiotin, and B

is a pyrimidine or 7-deazapurine.

These compounds can be prepared using a method comprising:

(a) to react a compound with the structure:

with a mercurial salt in a suitable solvent under suitable conditions so that a mercury compound is formed

with the structure:

(b) reacting said mercury compound with a chemical group which reacts with the -Hg<+> moiety in said mercury compound and represented by the formula ""'N, said reaction being carried out in an aqueous solvent and in the presence of f^PdCl ^under suitable conditions so that a connection is formed with the structure:

wherein N is a reactive functional end group or is A, and (c) recovering said compound as said modified nucleotide when N is A, or N is a reactive end group,

reacting said compound with a compound of the structure M-A, wherein M represents a functional group reactive with N in an aqueous solvent under suitable conditions, so that said modified nucleotide is formed, which is then recovered.

The present invention also provides compounds with the structure:

J

wherein each of B, B<1>and B" represents a purine, 7-deazapurine or pyrimidine moiety covalently bonded to the C11 position of the sugar moiety, provided that when B, B<1>or B"

is purine or 7-deazapurine, it is attached at the N 9 position of the purine or 7-deazapurine, and when B,B' or B" is pyrimidine, it is attached at the N^ position;

where A represents a moiety consisting of at least three carbon atoms capable of forming a detectable complex with

a polypeptide when the compound is introduced into a double-stranded duplex formed with a complementary ribonucleic or deoxyribonucleic acid molecule,

in which the dotted line represents a chemical bond joining B and A, provided that if B is purine the bond is attached to the 8-position of the purine, if B

is 7-deazapurine, the bond is attached to the 7-position of the deazapurine, and if B is pyrimidine, the bond is attached to the 5-position of the pyrimidine,

where z represents H- or HO- and

where m and n represent numbers from 0 up to approx. 100,000.

These compounds can be produced by enzymatic polymerization of a mixture of nucleotides comprising the modified nucleotides according to the invention. Alternatively, nucleotides contained in oligo- or polynucleotides can be modified using chemical methods.

Nucleotides which have been modified according to the present invention and oligo- and polynucleotides into which the modified nucleotides have been introduced, can be used as probes in biomedical research, clinical diagnosis and recombinant DNA technology. These different applications are based on the ability of the molecules to form stable complexes with polypeptides which in turn can be detected, either by means of the polypeptide's own properties or by means of detectable parts that are attached to, or that react with, the polypeptide.

Some applications include the detection and identification of nucleic acid-containing etiological agents, e.g. bacteria and viruses, secretion bacteria for antibiotic resistance, diagnosis of genetic diseases, e.g. thalassemia and sickle cell anaemia, chromosomal karyotyping and identification of tumor cells.

Several decisive criteria must be satisfied for a modified nucleotide to be generally suitable as a replacement

ning for a radioactively-labelled form of naturally occurring nucleotide. First, the modified compound must contain a substituent or probe that is unique,

i.e. not normally associated with nucleotides or polynucleotides. Second, the probe must react specifically with chemical or biological reagents to provide a sensitive detection system. Third, the analogs must be relatively efficient substrates for commonly investigated nucleic acid enzymes, since many practical applications require that the analog must be metabolized enzymatically, e.g.

must the analogues act as substrates for nucleic acid polymerase. To this end, the probe moieties must not be placed in ring positions that sterically or otherwise interfere with the normal Watson-Crick hydrogen bonding potential of the bases. Otherwise, the substituents will give compounds that are inactive as polymerase substrates. Substitution at ring positions that alter the normal "anti" nucleoside structure must also be avoided, since such structural changes usually render the nucleotide derivatives unacceptable as polymerase substrates. Normally, such considerations limit the substitution positions to the 5-position in a pyrimidine and the 7-position in a purine or a 7-deazapurine.

Fourth, the detection system must be able to react with the probe substituents introduced into both single-stranded and double-stranded polynucleotides in order to

be compatible with nucleic acid hybridization methods. To satisfy this criterion, it is preferred that the probe moiety is attached to the purine or pyrimidine by a chemical bond or "linker" so that it can readily react with antibodies, other detector proteins or chemical reagents.

Fifth, the physical and biochemical properties

to polynucleotides containing a small number of probe-

substituents are not significantly changed so that the current methods using radioactive hybridization probes do not need to be extensively modified. This criterion must be satisfied whether the probe is introduced by enzymatic or direct chemical methods.

Finally, the bond that attaches the probe part must resist

all experimental conditions to which normal nucleotides and polynucleotides are routinely subjected, e.g. extended hybridization times at elevated temperatures, extraction with phenol and organic solvents, electrophoresis, etc.

All these criteria are satisfied by the modified nucleotides described here.

These modified nucleotides have the structure:

where B represents a purine, 7-deazapurine or pyrimi-11

din moiety that is covalently bonded to the C -position of the sugar moiety, provided that when B is purine or 7-deazapurine, it is attached to the N° position of the purine or 7-deazapurine, and when B is pyrimidine, it is attached to N"*"-st ill none,

where A represents a moiety consisting of at least three carbon atoms capable of forming a detectable complex with a polypeptide when the compound is incorporated into a double-stranded ribonucleic acid, deoxyribonucleic acid duplex or a DNA-RNA hybrid,

where the dotted line represents a connecting group joining B and A, provided that if B is purine the bond is attached to the 8-position of the purine, if B

is 7-deazapurine, the bond is attached to the 7-position of the deazapurine, and if B is pyrimidine, the bond is attached to the 5-position of the pyrimidine, and

where each of x, y and z represents

These compounds are widely used as probes in biomedical research and recombinant DNA technology.

Although in principle all compounds covered by this structural formula can be prepared and used according to the practice of this invention, certain of the compounds are prepared or used more easily and are therefore preferred at present.

Thus, although purines, pyrimidines and 7-deazapurines are useful in principle, pyrimidines and 7-deazapurines are preferred since the purine substitution at the 8-position tends to render the nucleotides ineffective as polymerase substrates. Although thus modified purines are useful in some respects, they are not as generally useful as pyrimidines and 7-deazpurines. Moreover, pyrimidines and 7-deazapurines that can be used in the present invention must not be naturally substituted in the respective 5-

or the 7 positions. As a result of this, certain bases such as e.g. thymine, 5-methylcytosine and 5-hydroxymethyl-

cytosine not applicable. Bases currently preferred are cytosine, uracil, deazaadenine and deazaguanine.

A can be any moiety having at least three carbon atoms and capable of forming a detectable complex with a polypeptide when the modified nucleotide is incorporated into a double-stranded duplex containing either deoxyribonucleic or ribonucleic acid.

A can therefore be a single ligand having these properties, including haptens which are only immunogenic when attached to a suitable carrier, but are capable of reacting with suitable antibodies to form complexes. Examples of applicable parts include:

Of these, the preferred A moieties are biotin and iminobiotin.

Also, since aromatic moieties tend to intercalate into a base-paired helical structure, it is preferred that moieties A be non-aromatic. Since smaller parts also may not allow sufficient molecular reaction with polypeptides, it is preferred that A is at least C^, so that sufficient reaction can occur to

allow the formation of stable complexes. Biotin and iminobiotin satisfy both of these criteria.

The bond or group connecting part A to base B

may comprise any of the well-known bonds including carbon-carbon single bonds, carbon-carbon double bonds, carbon-nitrogen single bonds or carbon-oxygen single bonds. However, it is generally preferred that the chemical bond comprises an olefinic bond at the a-position relative to B. The presence of such an a-olefinic bond serves to keep the A moiety away from the base when the base pairs with another in the familiar double-helix configuration. This means that the reaction with the polypeptide occurs more easily, whereby the complex formation is made easier. Furthermore, single bonds with greater freedom of rotation cannot always keep the part sufficiently away from the helix to allow recognition of and complex formation with the polypeptide.

It is even more preferred that the chemical bonding group is obtained from a primary amine, and has the structure -Cr^-NH-,

since such bonds are readily formed using any of the well-known amine modification reactions. Examples of preferred bonds obtained from allylamine and allyl-(3-amino-2-hydroxy-1-propyl) ether groups have the formulas respectively

Although these linkages are preferred, others may be used, including in particular olefin linkages with other modifiable functionalities such as thiol-, carboxylic acid-

and epoxy functionalities.

The binding groups are attached in special positions, namely the 5-position in a pyrimidine, the 8-position in a pyrin or the 7-position in a deazapurine. As indicated previously, substitution at the 8-position of a purine does not provide a modified nucleotide that is useful in all of the methods discussed herein. It could be that the 7-position in a purine, which is occupied by a nitrogen atom, could be the point of attachment. However, the chemical substitution methods currently used and discussed here are not suitable for this purpose.

The letters x, y and z represent groups attached at the 5'-3' and 2' positions of the sugar moiety. They can be any of

Although conceivable, it is unlikely that both x, y and

z at the same time will be the same. It is more likely that at least one of x, y and z will be a phosphate-containing group,

either mono-, di- or tri-phosphate and that at least one will be Ho- or H-. As will be readily understood, the most likely identity of z will be HO- or H- indicating ribonucleotide or deoxyribonucleotide respectively. Examples of such nucleotides include 5<1->ribonucleoside monophosphates, =<1->ribonucleoside diphosphates, 5<1->ribonucleoside triphosphates, 5<1->deoxyribonucleoside monophosphates, 5'-deoxyribonucleoside diphosphates, 5<1->deoxyribonucleoside triphosphates, 5'p-ribonucleoside-3'p. and 5'p-deoxyribonucleoside3<1>p. More particular examples include modified nucleotides

of this type in which A is biotin or iminobiotin, the chemical bond being

and B is uracil or cytosine.

The general synthetic procedure used to introduce the linker arm and probe portion onto the base is discussed above. (See especially, J.L. Ruth and D.E. Bergstrom, J.Org. Chem., 4_3'2870, 1978, D. E. Bergstrom and M.K.Ogawa, J.Amer.Chem.Soc.100, 8106, 1978 and C.F.Bigge, P.Kalaritis, J. R. Deck and M. P. Mertes, J. Amer. Chem. Soc. 102, 2033, 1980.). However, the olefin substituents used here are

not previously used. In order to make the attachment of the probe part A easier, it has been found particularly desirable to use olefins with primary amine functional groups, such as e.g. allylamine [AA] or allyl-(3-amino-2-hydroxy-1-propyl) ether [NAGE], which allow probe attachment by means of standard amine modification reactions, such as

Due to the ease of preparation, it is preferred to use NHS esters for probe addition. However, olefin binding arms with other modifiable functional groups can also be used, such as e.g. thiols, carboxylic acids, epoxides and the like. Furthermore, both tie arm and probe can be added in a single step if desired.

In particular, modified nucleotides with the structure can be produced:

wherein B represents a purine, 7-deazapurine, or pyrimidine moiety covalently attached to the C 1 • position of the sugar moiety, provided that when B is purine or 7-deazapurine, it is attached at the N 9 position of the purine or deazapurine , and when B is pyrimidine, it is attached in the N^ position,

where A represents a moiety consisting of at least three carbon atoms capable of forming a detectable complex with its polypeptide when the compound is introduced into a double-stranded ribonucleic acid, doxyribonucleic acid duplex, DNA-RNA hybrid,

where the dotted line represents a chemical bond joining B and A, provided that if B is purine, the bond is attached at the 8-position of the purine, if B is 7-deazapurine, the bond is attached at the 7-position of the deaza-

the purine, and if B is pyrimidine, the bond is attached in the 5-position of the pyrimidine, and

where each of x, y and z represents

in that:

(a) a connection with the structure

is reacted with a mercurial salt in a suitable solvent under suitable conditions, so that a mercuric compound is formed with the structure: (b) said mercuric compound is reacted with a chemical moiety which is reactive with the -Hg<+> moiety in said mercuric compound and represented by the formula * *'N, said reaction being carried out in an aqueous solvent and in the presence of I^PdCl^ under suitable conditions so as to form a compound with the structure

wherein N is a reactive functional end group or is A, and (c) said compound is recovered as said modified nucleotide when N is A, or when N is a reactive end group, said compound is reacted with a compound of the structure M-A, wherein M represents a functional group reactive with N in an aqueous solvent under suitable conditions, so that said modified nucleotide is formed, which is then recovered.

The following form is illustrative:

Although the reactions can be carried out at hydrogen ion concentrations as low as pH 1, or as high as pH 14, it is preferred to work in the range from approx. 4 to approx. 8. This is especially the case when working with unstable connections such as e.g. nucleoside polyphosphates, polynucleotides and nucleo-

time coenzymes that are hydrolysed at pH values outside this range. Likewise, it is preferred to work at a temperature in the area from approx. 20 to 30°C to avoid possible decomposition of labile organic substrates. The reactions can, however, be carried out at temperatures from approx. 5 to 100°C.

As is common with chemical reactions, higher temperatures promote the reaction rate and lower temperatures slow it down. In the temperature range from 5 to 100°C, the optimal reaction time can thus vary from approx. 10 minutes to 98 hours. In the preferred temperature range, the reaction times normally vary from approx. 3 to approx. 24 hours.

The preferred method of maintaining the pH in the desired range is to use buffers. A variety of buffers can be used. These include e.g. sodium or potassium acetate, sodium or potassium citrate, potassium citrate phosphate, tris acetate and borate sodium hydroxide buffers. The buffer concentration can vary within a wide range, up to approx. 2.0 molar.

While a particular advantage of mercuration and palladium-catalyzed addition reactions is that they can be carried out in water, small amounts of an organic solvent can advantageously be mixed in, such as a solubility reducer<p>. The organic solvents usually chosen are those which are miscible with water. These can be chosen from ethers, alcohols, esters, ketones, amides and the like such as e.g. methanol, ethanol, propanol, glycerol, dioxane, acetone, pyridine and dimethylformamide. However, since it has been observed that the presence of alcohols, such as methanol, often resulting in alkoxy addition across the olefin double bond, must

any organic solvent used as a solubility aid is carefully chosen. Introduction of alkoxy substituents to the exocyclic α- or 3-carbon atoms often results in the production of compounds that are less effective for use as enzyme substrates.

Although various mercuric salts may be used, mercuric acetate is the currently preferred salt. As indicated earlier, the connections can also be made by first adding a tie arm and then part A, or by adding a tie arm to which A is already attached.

Thus, the chemical part represented by the formula ••*N can be any of the many groups that finally result in the production of the desired compounds.

Examples include -CH=CH-CH2~NH2,

-CH=CH-CH2-0-CH2-CH-CH2-NH2, -CH=CH-CH2-NH-biotin and

OH

-CH=CH2-CH2-0-CH2-CH-CH2-NH-iminobiotin.

OH

The amounts of the reactants used in these reactions

can vary greatly. In general, however, the amounts of non-mercury compounds, mercury compound and palladium-containing compound will be essentially stoichiometric, while the mercury salt and the compound <*>'"N will be present in molar excess, e.g. 5-20 mol • • *N or mercurial per mole of mercury compound or non-mercury compound, respectively.In practice, the amounts will vary depending on variations in the reaction conditions and the exact identity of the reactants.

Having a biotin probe directly attached to nucleotide derivatives that are capable of acting as enzyme substrates provides great versatility, both in the experimental protocols that can be performed and in the detection methods (microscopic and non-microscopic) that can be used for analysis. Biotin nucleotides can, for example, be introduced into polynucleotides that are just synthesized by cells or crude cell extracts, which makes it possible to detect and/or isolate growing polynucleotide chains. Such a method is impossible to carry out by a direct chemical modification method. Furthermore, enzymes can be used as reagents for introduction

of probes, such as biotin, in highly selective or position-specific sites in polynucleotides. The chemical synthesis of similar probe-modified products would be very difficult to achieve at best.

The synthesis of nucleotides containing biotin or iminobiotin was accomplished as detailed in the examples set forth below. Pyrimidine nucleoside triphosphates containing one of these probes attached to the C-5 carbon atom were good to excellent substrates for a wide variety of purified nucleic acid polymerases of both prokaryotic and eukaryotic origin. These include DNA polymerase I from E. coli, bacteriophage T4 DNA polymerase, DNA polymerases a and 3 from murine (A-9 and human (HeLa) cells of Herpes simplex virus DNA polymerase. Confirmatory data were obtained with E. coli DNA polymerase I using either the cut-transfer condition of R;'„gby, et al. (P.W.J. Rigby, M.Dieckmann, C. Rhodes and P. Berg, J. Mol.Biol. 113 , 237, 1977) or the gap-filling reaction described by Bourguignon et al. (G.J. Bourguignon, P.J. Tattersall and D.C. Ward, J.Virol. 20, 290, 1976). Bio-dUTP has also been found to

act as a polymerase substrate both in CHO cells, which are permeabilized by treatment with lysolecithin according to the method of Miller, et al. (M.R. Miller, J.C. Castellot,

Jr. and A.B. Pardee, Exp. Cell Res. 120, 421, 1979) and in a nuclear reproduction system prepared from Herpes simplex-infected BHK cells. Although biotinyl ribonucleoside triphosphates were found to function as substrates for . RNA polymerase of Ecoli and bacteriophage T7, they are used

not as effective as their deoxyribonucleotide triphosphate counterparts. Indeed, they are introduced poorly if at all by the eukaryotic RNA polymerases that have been examined

(HeLa cell RNA polymerase III, Calf thymus RNA polymerase

II and mouse cell RNA polymerase II). While this limited range of substrate function limits applicability in some in vivo or in vitro replication assays, biotin-labeled RNA probes can be enzymatically prepared from DNA templates using E. coli or T7 RNA polymerases or by 3' end-labeling methods using of RNA ligase with compounds such as e.g. biotinyl-pCp. However, AA and NAGE derivatives of UTP are substrates for the eukaryotic RNA polymerases mentioned above. With the availability of antibodies to these analogs, the isolation of growing mimics by immunological or affinity methods should be possible.

The enzymatic polymerization of nucleotides in-

containing biotin or iminobiotin substituents were not monitored directly, as none of these probes were radiolabelled. However, two series of experiments clearly show that biotinyl nucleotides were introduced. The first is that polynucleotides that were synthesized in the presence of biotin nucleotides are selectively retained when chromatographed over avidin or streptavidin affinity columns.

(Tables I and II). While normal DNA, which has for example been sectioned with 3 2 P-dAMP, is eluted quantitatively with the addition of 0.5 M NaCl, the main amount of biotinyl-DNA or iminobiotinyl-DNA remains bound to the resin even after extensive washing with strong salt solution, urea, guanidine- HCl, formamide. or 50 mM NaOH. The small fraction of radiolabel eluted using these washing conditions is not retained when applied to the resin the second time, suggesting that the radioactivity is associated with DNA fragments free of biotin substitution. The second line of evidence is that only biotin-labeled polynucleotides are immunoprecipitated when treated with purified anti-biotin IgG followed by formalin-fixed Staphylococcus aureus. (Table III). It is clear from the data in these tables that extremely small amounts of biotin can be detected by this method. These results also show that the biotin molecules can be detected by avidin, streptavidin or specific antibodies while the DNA is still in its native, double-stranded form, a condition that is absolutely essential if the anti-body binding or the avidin affinity experiments are to be applicable in probe detection using hybridization techniques.

It is thus possible to produce new compounds with the structure:

wherein each of B, B' and B" represents a purine, deaza-

1' purine or pyrimidine part which is covalently bound to the C position in the sugar part, provided that when B, B', or B"

is purine or 7-deazapurine, it is attached at the N 9 position of the purine or deazapurine, and when B,B' or B" is pyrimidine, it is attached at the N^ position,

where A represents a moiety consisting of at least three carbon atoms capable of forming a detectable complex with a polypeptide when the compound is introduced into a double-stranded duplex formed with a complementary ribonucleic or deoxyribonucleic acid molecule,

in which the dotted line represents a linking group joining B and A, provided that if B is purine the bond is attached to the 8-position of the purine, if B is 7-deazapurine, the bond is attached to the 7-position of the deazapurine and if B is pyrimidine, the bond is attached to the 5-position of the pyrimidine,

where z represents H- or HO-, and

where m and n represent numbers from 0 up to approx. 100,000.

It is of course easy to understand that, in general, m and

n at the same time be 0 since the compound in that case only becomes a modified nucleotide as described earlier. In general, B' and B" will vary within the same oligo- or poly-nucleotide, and are alternatively uracil, cytosine, thymine, guanine, adenine or the like. In general, the variation will also correspond to the ordered sequence of nucleotides that code for the synthesis of peptides according to the well-known Genetic Code. However, it is intended that the shown structure should also include polynucleotides such as poly C, poly U, poly r(A-U and poly d(A-U) as well as calf thymus DNA, ribosomal RNA of E. coli or yeast, bacteriophage RNA and DNA (R17, fd), animal virus (SV40 DNA), chromosomal DNA, and the like, provided

only that the polynucleotides are modified according to the present invention.

It should also be understood that the structure comprises more than one modified nucleotide in the oligomer or polymer, e.g. from two to thirty modified nucleotides. The critical factor in this regard is that the number of modifications should not be so large that the polynucleotide is rendered ineffective for the intended application.

Finally, it should be understood that modified oligo- and polynucleotides can be combined to form larger units with the same structure as long as the end groups are made compatible or reactive.

These compounds can be prepared by enzymatic polymerization of suitable nucleotides, especially nucleotide triphosphates in the presence of a nucleic acid template which directs the synthesis under suitable conditions. Such conditions can vary greatly depending on the enzyme used, the amounts of nucleotides present and other variables. Illustrative enzymes include E. coli DNA polymerase I, bacteriophage T 4 DNA polymerase, DNA polymerases a and 3 from murine and human (HeLa) cells, Herpes simplex virus DNA polymerase, E. coli RNA polymerase , RNA polymerase of bacteriophage T7, eukaryotic RNA polymerase including HeLa cell RNA polymerase III, calf thymus RNA polymerase II and mouse cell RNA polymerase II.

The compounds can also be produced by end addition to oligo- or poly-nucleotides to produce compounds where m or n is 0 depending on whether the addition takes place in the 5'- or 3<1-> position. Furthermore, connections such as e.g. pCp or pUp in which the base is biotinized is added to existing molecules using the enzyme RNA ligase.

Modified oligo- and polynucleotides can also be produced by chemical modification of existing oligo- or polynucleotides using the procedure described earlier for modification of individual nucleotides.

The various modified nucleotides, oligonucleotides

and the polynucleotides of the invention can<p>be detected by bringing the compounds into contact with the polypeptides capable of forming complexes with them under suitable conditions so that complexes are formed, provided that the polypeptides comprise one or more moieties that can be detected when the complex or complexes are formed>generally using conventional detection techniques.

A polypeptide detector for the fen probe of the biotinyl type is avidin. The reaction between avidin and biotin exhibits one of the tightest non-covalent bond constants (K .,. =10-"1" )

^ dis

that are found in nature. If avidin is linked with potentially detectable indicator molecules, e.g. fluorescent dyes (fluoroscein, rhodamine), electron-dense reagents (ferritin, hemocyanin, colloidal gold) or enzymes

which is able to precipitate insoluble reaction products (peroxidase, alkaline phosphatase) the presence, location and/or quantity of the biotin probe can be determined.

Avidin unfortunately has a property that makes it less desirable as a biotin indicator protein when used in conjunction with nucleic acids or chromatin materials.

It has been reported (M.H. Heggeness, Stain Technol. , 5_2, 165, 1977, M.H. Heggeness and J.F. Ash, J. Cell. Biol., JA'783>1977, E.A. Bayer and M. Wilchek, Methods of Biochemical Analysis 26, 1 , 1980) that avidin binds firmly to condensed chromatin or to subcellular fractions containing large amounts of nucleic acid in a manner independent of its biotin-binding ability. Since avidin is a basic glycoprotein with a pl of 10.5, its histone-like character or its carbohydrate moieties are most likely

responsible for these observed non-specific reactions.

A preferred probe for biotin-containing nucleotides and derivatives is streptavidin, an avidin-like protein synthesized by the soil organism Streptomyces avidinii. Its preparation and purification are described in Hoffman, et al., Proe. Nati. Acad. Sci., 7J_, 4666 (1980). Streptavidin has

a much lower pI (5.0), is non-glycosylated and shows much lower non-specific binding to DNA than avidin and therefore has potential advantages when used as

understands methods for nucleic acid detection.

The most preferred protein for biotin-like probe detection is monospecific rabbit IgG, antibiotin immunoglobulin. This compound was produced by immunization of rabbits

with bovine serum albumin conjugated biotin as described previously.

(M. Berger, Methods in Enzymology, 6_2, 319 (1979)) and purified by affinity chromatography. Although the association constant of immunoglobulin haptens has values of Kassn (10 to

-ji^q assn

10 ) which are significantly lower than for avidin-biotin complexes, they are essentially equivalent to those observed with the avidin-iminobiotin complex. Furthermore, the anti-biotin antibodies have proven very useful for the detection of specific polynucleotide sequences on chromo-

which by in situ hybridization since little, if any, non-specific binding of the antibody to the chromatin material occurs.

The modified polynucleotides according to the invention are capable of denaturation and renaturation under conditions compatible with their use as hybridization probes. An analysis of the heat denaturation profiles and hybridization properties of several biotin-substituted DNA and RNA polymers clearly indicates this. For example, pBR 322 has DNA or X^DNA, which is cross-transferred to introduce approx. 10-100 biotin residues per kilobase, Tm values essentially identical to those of the control, biotin-free DNA. Furthermore, the 32p-labeled, biotin-substituted, pBR 322 DNA exhibits the same degree of specificity and autoradiographic signal intensity as the control, thymidine-containing DNA, when used as a hybridization probe for the detection of bacterial colonies containing the plasmid.

In DNA duplexes, such as MVM RF DNA, in which each thymidine residue in one strand (1250 total per 5 Kb) is replaced with a biotinyl nucleotide, the Tm is only 5°C less than that of the unsubstituted control. Although the Tm of poly d(A-bicU) in which each base pair contains a bio-dUMP residue is 15°C lower than the poly d(A-T) control, the degree of cooperativity and degree of hyperchromicity observed both during denaturation and renaturation the same for the two polymers. A parallel analysis of RNA duplex and DNA/RNA hybrids indicates that their Tm also decreases as the biotin content of the polymer increases. However, it is clear that a significant number of biotin molecules can be introduced without significantly changing the hybridization properties of the polymers.

These results strongly suggest that biotin-substituted polynucleotides can be used as probes for the detection and/or localization of specific polynucleotide sequences in chromosomes, fixed cells, or tissue sections. The general protocol for detection of the biotin-substituted probe is schematically illustrated as follows: This general schematic only illustrates methods used for gene mapping (cytogenetics), and recombinant DNA technologies. However, it can equally well be used for the detection of nucleic acid sequences of bacterial, viral, fungal or parasitic origin in clinical samples and this forms the basis for an important new method for clinical diagnostics that is not based on the use of radioisotopes.

Immunological and histochemical methods for the detection of biotin have shown that the basic procedure is applicable for rapid methods of gene mapping by in situ hybridization and non-radioactive methods for the detection of specific nucleic acid sequences by means of spot hybridization methods. This technology can be used in the development of new clinical diagnostic procedures.

Using this method, it is possible to determine the presence of a specific deoxyribonucleic or ribonucleic acid molecule, particularly such a molecule obtained from a living organism, e.g. bacteria, fungi, viruses, yeast or mammals. This in turn allows the diagnosis of nucleic acid-containing etiological agents in a patient or another being.

Moreover, it provides a method for screening bacteria to determine antibiotic resistance. Thus, for example, penicillin resistance can be determined in Streptococcus pyogenes or Neisseris meningitidis, tetracycline resistance in Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Streptococcus pyogenes or Neisseria gonorrhoeae and aminoglycoside resistance in Mycobacterium tuberculosis.

In these methods, a polynucleotide is produced which is complementary to the nucleic acid sequence which characterizes the organism or its antibiotic resistance and which additionally comprises one or more modified nucleotides according to the invention. This polynucleotide is hybridized with nucleic acid obtained from the organism being examined. Failure in terms of hybridization indicates absence of the organism or of resistance characteristics. Hybridized nucleic acid duplexes are then identified by forming a complex between the duplex and an appropriate polypeptide, carrying a detectable moiety, and detecting the presence of the complex using an appropriate detection technique. Positive detection indicates that the complex, the duplex and therefore the relevant nucleic acid sequence is present.

This method can be extended to the diagnosis of genetic diseases, such as e.g. thalassemia and sickle cell anemia. The deoxyribonucleotide gene sequence whose presence or absence (in the case of thalassemia) is associated with the disease can be detected by following hybridization with a polynucleotide probe according to the invention, based on complex formation with a suitable detectable polypeptide.

Mapping genes or their mimics to specific locations on chromosomes has been a far-reaching and time-consuming task, which mainly includes techniques with cell fusion and somatic cell genetics. Although in situ hybridization has been successfully used for mapping single copies of gene sequences in species undergoing chromosome polytenization, such as e.g. In Drosophila, detection of genes with particular sequences in most higher eukaryotic chromosomes has been very difficult, if not impossible, using standard hybridization methods. The necessity of polynucleotide probes with very high specific radioactivity to facilitate autoradiographic localization of hybridization sites results in rapid radiodegradation of the probe and an accompanying increase in the background noise of the silver grain deposit. The use of hybridization probes with low to moderate specific radioactivities requires exposure times of many days or weeks, even to detect multicopy sequences, such as ribosomal RNA genes or satellite DNA. Since recombinant DNA technology has made possible the molecular cloning of essentially any single-copy sequence found in eukaryotic cells, it would be very useful to have a rapid and sensitive method for mapping the chromosomal origin of such cloned genomic fragments.

Modified nucleotides can be used in methods for gene mapping by means of in situ hybridization which bypasses the use of radioisotopes. This method uses a thymidine analogue containing biotin which can be introduced enzymatically into DNA probes by section transfer. After hybridization in situ, the botin molecules serve as antigens for affinity-purified rabbit anti-biotin antibodies. Immunofluorescent antibody sandwiches prepared with fluorescein-labeled goat anti-rabbit IgG enable rapid and specific cytogenetic localization of cloned gene sequences as green-yellow bands. This method has four main advantages compared to conventional autoradiographic methods of in situ gene localization, less background noise, an increase in resolving power between bands, a reduction in the time required to determine the position of probe hybridization, and chemically stable hybridization probes.

This method has been used successfully for localization

of repeated and individual DNA sequences in the polytene chromosome of Drosophila milanogaster and satellite DNA on mouse metaphase chromosomes.

It has thus been found that polytene chromosomes can be used as a test system for determining the effectiveness of probes using the modified nucleotides according to the present invention as demonstrated by indirect immunofluorescence for gene mapping in situ. The probes included a number of cloned Drosophila sequences obtained from Otto Schmidt and Dieter Soll, which e.g. tRNA genes cloned into plasmid vectors with inserts with sizes in the range from approx.

5 to approx. 22 kilobases. Many of these clones have already been assigned to particular bands on the chromosome map of Drosophila by means of conventional in situ hybridization methods using radioisotopes.

DNA probes were nicked in the presence of Bio-dUTP. Off and on

until 3 H dATP and/or<3>H dCTP were introduced into the slice transfer reaction mixture. This enabled both autoradiographic and immunofluorescent localization of a sequence on a single chromosome spread. In situ hybridization was performed as described in M.L. Pardue, and J.G. Gall, Methods in Cell Biol., 10, 1 (1975). After the final 2 x SSC wash to remove unhybridized probe, the slides were cleaned with PBS (phosphate buffered saline) and incubated at 37°C with 2.5 µg/ml rabbit anti-biotin in PBS and 10 mg/ml BSA for 2-16 hours. This was followed by incubation of the slides with FITC-labeled goat anti-rabbit IgG (Miles Laboratories, diluted 1:100 in PBS and 10 mg/ml BSA) for one to four hours. Evans blue was often required as a red counterstain to view the chromosomes with fluorescent illumination.

When plasmids pBR 17D and pPW 539 containing 5 Kb and 22 Kb inserts were hybridized using this method, it was found that the hybridization pattern is reproducible from spread to spread and is unequivocally observed on

more than 90% of the chromosome spreads on a given slide.

The cloned transposable element pAC 104 is known to fit into many places along the Drosophila genome. Comparison between the autoradiogram and the fluorescent image obtained by in situ hybridization of this probe illustrates a main advantage of this method, i.e. that where diffuse regions of silver grains appear on an autoradiogram, duplicates or a series of bands can be distinguished by immunofluorescent labeling.

The other immediately obvious advantage of this method is the considerably less time required for gene designation by indirect immunofluorescence. A designation of a DNA fragment to a particular band within six hours of hybridization.

This can be compared to days or weeks required

for autoradiographic exposure methods. This factor, in combination with increased resolution, makes the use of modified nucleotides detected by indirect immunofluorescence immediately preferable to the more classical methods.

It has been shown that this immunological method can also be carried out with mammalian chromosomes in which satellite DNA has been found to lie in the geometric center of gravity areas in the mouse. metaphase chromosomes. The results form a basis for the development of a simple gene mapping procedure for single copy sequences in chromosomes from humans and other mammals. Such a procedure will greatly facilitate our understanding of the genetic organization in the chromosomes and make clinical cytogenetic diagnoses much faster and more practical.

While a single-step "antibody sandwich" method, in which the chromosome spread is examined, post-hybridization, with rabbit anti-biotin IgG can be covered, this protocol may not generate sufficient fluorescence for unequivocal gene designation. However, a much stronger fluorometric signal can be obtained using the "hapten-antibody sandwich technique" described by Lamm, et al. (1972),

Wofsy, et al., (1)74). In this method, the primary antibody, in our case monospecific, is modified

rabbit anti-biotin IgG, chemically with a haptenizing reagent, such as 2,4-dinitrofluorobenzene, preferably while the immunoglobulin is bound to an antigen affinity column (biotin-Sepharose TM). As many as 15-20 hapten (DNP) groups can be attached to the primary antibody without diminishing its antigen-binding affinity or specificity (Wallace and Wofsy, 1979). If the primary antibody treatment

of the sample is followed by an incubation with a fluorescently labeled anti-hapten IgG antibody, instead of a fluorescently

end labeled anti-IgG, a 5-7-fold increase in fluorescence signal can be achieved. Since monospecific guinea pig anti-DNP IgG is also available, this secondary antibody can be haptenized with biotin and thus generate two anti-hapten IgG populations, DNP-labeled anti-biotin IgG

and biotin-labeled anti-DNP IgG. If these can be used alternately to achieve several rounds of hapten-antibody sandwich formation and then followed by fluorescently labeled protein A from Staphylococcus aureus, which binds specifically to IgG molecules from many mammalian species,

it could result in an enormous amplification of the primary antibody signals with subsequent applicability.

The protein streptavidin from Streptomyces avidini is a potential alternative to anti-biotin IgG as an aid for species-specific targeting of a coupled display system [e.g. fluorescent probes (above) or histochemical reagents (below)] to the site of the hybridized biotin-containing polynucleotide. One of the advantages of streptavidin over anti-biotin IgG is that its affinity for biotin is Kassn=10 15 m7enassociation constants for hapten-IgG reactions are 10 to 10 . The high reaction rate and extreme affinity mean that the time required to localize the biotinized probe will be minutes with streptavidin versus hours with immunological reagents.

Initial evaluations of a streptavidin detection system are in progress. Polytene chromosomes hybridized with biotinized DNA probes will be incubated with streptavidin followed by a subsequent incubation with bovine serum albumin, which is dual-labeled with biotin and FITC (FITC, biotinyl-BSA). Since only one of the four streptavidin subunits is believed to be involved in binding at each biotinylated DNA site, potentially a labeled BSA molecule could bind to each of the remaining three unconjugated subunits of the streptavidin-biotinyl-nucleotide complex. The fluorescence signal from this single streptavidin + FITC, biotinylBSA layer will be compared to a control using the basic "antibody sandwich method" described previously.

If "antibody-Sandwich" and streptavidin + FITC, biotinyl-BSA detection intensities are comparable, attempts can be made to extend the streptavidin + FITC, biotinyl-BSA system to single-copy copy sensitivity in a manner parallel to the multiple "hapten -antibody-sandwich" method.

Since some of the biotin groups on BSA will not be bound to the first layer of streptavidin, a second layer of streptavidin can be added until a sufficient signal is obtained. If, for example, in the second layer only two streptavidin protomers are bound to each first layer of BSA and each of these streptavidin protomers is bound three FITC-biotinyl BSA molecules, then the intensity in the second layer will be twice as great as that from the first layer, for the third layer, with analogous binding stoichiometries, the fluorescence intensity will be 12 times as

large as in the first layer, so that the total intensity will quickly increase with successively added layers.

There are plans to use a larger carrier protein such as thyroglobulin instead of BSA to maximize the amounts of attached fluorescence and biotin probes. It may also be necessary to use a longer linker arm between the biotin probe and the carrier protein. A longer linker arm should sterically optimize the theoretical delivery of a biotinized fluorescent carrier molecule to each unconjugated streptavidin subunit and maximize the number of streptavidin protomers in the subsequent layer that will bind to the biotinized fluorescent carrier. As before, appropriate controls will be performed to ensure that substitution of the carrier protein with fluorescent probes and biotin does not cause solubility and/or non-specific binding problems. The streptavidin carrier protein delivery system has two significant advantages over the immunofluorescent method in addition to its delivery rate. First, only two protein components are needed to

form the layers. Second, only the carrier protein needs to be modified and there is no need to maintain functional or even total structural integrity as long as the biotin groups are accessible to streptavidin.

An alternative to the fluorescence method for visualizing hybridized probes is to direct enzymes such as peroxidase, alkaline phosphatase of 3-galactosidase to the hybridization site where enzymatic conversion of soluble substrates to insoluble colored precipitates enables light microscopic visualization. The major advantage of this technique is that the histochemical methods are 10 to 100 times as sensitive as fluorescence detection. In addition, the colored precipitates do not fade with too much light exposure and thus avoid one of the main disadvantages of fluorescent light microscopy. These enzymes can be linked to the final antibody instead of fluorescent probes in the "hapten-antibody sandwich" technique using bifunctional reagents such as glutaraldehyde or in the case of peroxidase via oxidation of the peroxidase carbohydrate parts to aldehydes and coupling of these residues with ε-amino groups in the desired protein. For the streptavidin-biotinized carrier protein method, an enzyme with attached biotinyl groups could replace a fluorescence-biotinized carrier system. Alternatively, the enzyme could be linked via biotin to the last layer of streptavidin, as amplification of streptavidin sites is built up in the previous layers using biotinised BSA or thyroglobulin. In the near future, the necessary histochemical reagents and the appropriate substrate/insoluble product combinations will be developed for visualization of in situ hybridizations without background problems. The histochemical methods of signal amplification

should therefore be ready for trials in the summer of 1981.

Detection and/or imaging of very low levels of fluorescent light is possible using image intensifiers or systems that have been available in the past consisting of lasers and photomultipliers. These methods allow the detection of light down to the level of single photons. With suitable digital processing systems, images can be produced in which each point in the image is strictly proportional to the number of photons emitted from a point on the object. By using systems of this kind or flow systems in which the cells or parts of the parts rush past a laser beam, increases in the detection sensitivity for fluorescent materials by factors between 100 and 1000 beyond what can be detected with the eye can be achieved. This increase is sufficient to detect the fluorescence from single copy genes.

In a preferred modification, analogues have been synthesized

of dUTP and UTP containing a biotin molecule covalently bound to the C-5 position of the pyrimidine ring by means of an allylamine linker. These biotinyl nucleotides are efficient substrates for a variety of DNA and RNA polymerases in vitro. DNA containing low levels of biotin substitution (50 molecules or less/kilobases)

has denaturation, reassociation and hybridization properties that are indistinguishable from those of unsubstituted control DNA.

Thus, the present invention also provides a method for chromosome karyotyping. In this method, modified polynucleotides are prepared that correspond to known genes and include modified nucleotides. Certain polynucleotides are hybridized with chromosomal deoxyribonucleic acid and the resulting duplexes are brought into contact with appropriate polypeptides under appropriate conditions to allow complex formation. The polypeptides comprise detectable portions so that the location of the complexes can be determined and the location of particular genes thereby fixed.

Another embodiment of the present invention comprises the detection of poly A-containing sequences using poly U in which some of the uracil bases are modified so that they contain a probe. Yet another embodiment comprises cyclically modified nucleotides in which two of x, y and z are reacted to form the cyclic moiety

Such cyclically modified nucleotides can then be used for

to identify hormone receptor sites on the cell surfaces which in turn can be used as a method to detect cancer or tumor cells.

Finally, tumor cells can be diagnosed by producing polynucleotides that are modified according to the present invention and are complementary to the precursor ribonucleic acid that is synthesized from a deoxyribonuclein

acid gene sequence, which is associated with the production of polypeptides, such as e.g. α-fetal protein or carcinoembryonic antigen, the presence of which is diagnostic of specific tumor cells. Hybridization and detection of hybrid du-

plexes would thus constitute a step forward for detecting the tumor cells.

The examples that follow are given to illustrate various aspects of the present invention, but are not intended to limit its scope in any way as is more particularly stated in the claims.

Examples 1 and 2

Synthesis of biotinyl - UTP and biotinyl - dUTP

a) Preparation of labeled nucleotides

UTP (570 mg, 1.0 mmol) or dUTP 554 mg, 1.0 mmol) were

dissolved in 100 ml of 0.1 M sodium acetate buffer pH 6.0 and mercuric acetate (1.59 gm, 5.0 mmol) added. The solution was heated to 50°C for 4 hours, and then cooled on ice. Lithium chloride (392 mg 9.0 mmol) was added and the solution extracted six times with an equal volume of ethyl acetate to remove excess HgCl 2 . The efficiency of the extraction process was checked by determining the mercury ion concentration in the organic layer using 4,4'-bis(dimethylamino)-thiobenzophenone (A.N. Cristoper, Analyst, 94_, 392 (1969). The degree of nucleotide mercuration, determined spectrophotometrically after iodination of an aliquot of the aqueous solution as described by Dale et al (R.M.K. Dale, D.C. Ward, D.C. Livingston, and E. Martin, Nucleic

Acid Res. 2, 915 [1975]), was routinely between 90 and 100%. The nucleotide products in the aqueous layer, which often became cloudy during the ethyl acetate extraction, were precipitated by the addition of three volumes of ice-cold ethanol and collected by centrifugation. The precipitate was washed twice with cold absolute ethanol, once with ethyl ether and then air dried. The mercuriated nucleotides thus prepared were used for the synthesis of allylamine nucleotides without further purification.

b) Synthesis of allylamine - dUTP and allylamine - UTP

The mercuriated nucleotides (from tin a) were dissolved in

0.1 M sodium acetate buffer at pH 5.0 and adjusted to a concentration of 20 mM (200 OD/ml at 267 nm). A new 2.0

M solution of allylamine acetate in aqueous acetic acid was prepared by slowly adding 1.5 ml of allylamine (13.3 mmol) to 8.5 ml of ice-cold 4 M acetic acid. 3 ml (6.0 mmol) of the neutralized allylamine stock solution was added to 25 ml (0.5 mmol) of the nucleotide solution. A nucleotide equivalent of ^PdCl^, (163 mg, 0.5 mmol) dissolved in 4 mL of water was then added to initiate the reaction. On addition of the palladium salt (Alfa-Ventron), the solution gradually became black from the metal (Hg and Pd) deposit which appeared on the walls of the reaction vessel. After standing at room temperature for 18-24 hours, the reaction mixture was passed through a 0.45 mm membrane filter (Nalgene) to remove most of the remaining metal precipitate. The yellow filtrate was diluted five times and applied to a 100 ml column of DEAE-"Sephadex" TM A-25 (Pharmacia). After washing with one column volume of 0.1 M sodium acetate buffer at pH 5.0, the products were eluted using a one liter linear gradient (0.1-0.6M) of either sodium acetate at pH~'8-9,

or triethylammonium bicarbonate (TEB) at pH 7.5. The desired product was in the largest UV-absorbing fraction eluted between 0.3 and 0.35 M salt. Spectral analysis showed that this peak contained several products, and final purification was achieved by reverse phase - HPLC chromatography

on columns of "Patisil" - ODS 2, using either 0.5 M NH^H^O^ buffer at pH 3.3 (analytical separations), or 0.5 M triethylammonium acetate at pH 4.3 (preparative separations) as eluents. 5<1->triphosphates of 5-(3-aminopropen-1-yl)uridine (allylamine adduct of uridine) were the last moieties eluted from the HPLC column and they were clearly separated from three uncharacterized impurities. These nucleotides were characterized by proton NMR elemental analysis [AA-dUT<P>(<C>12 H16 N3<0>14 P^a^1H20): theory C, 22.91, H, 2.88, N, 6.68 , P, 14.77, Found, C, 23.10,

H, 2.85, N, 6.49, P, 14.75.

AA-UT<P>(C12 HlgN3<0>15P3 Na4' 4H20): Theory, C 20.61,

H, 3.46, N, 6.01, P, 13.3. Found C, 20.67, H, 4.11,

N, 5.39, P, 13.54] spectrally and chromatographically.

c) Biotinization of AA-dUTP or AA-UTP

Biotinyl N-hydroxysuccinic acid ester (NHSB) was prepared from

biotin (Sigma) as described previously (H. Heitzmann and F.M. Richards, Proe, Nat. Acad. Sei. USA. 71, 3537

[1974]). AA-dUTP<*>H2O (63 mg, 0.1 mmol) or AA-UTP'4H2O

(70 mg, 0.1 mmol) was dissolved in 30 mL of 0.1 M sodium borate buffer at pH 8.5 and NHSB (34.1 mg, 0.1 mmol) dissolved in 2 mL of dimethylformamide was added. The reaction mixture

allowed to stand at room temperature for 4 hours and then passed directly onto a 30 ml column of DEAE-"Sephadex" TM A-25, which had previously been brought to equilibrium with 0.1 M TEAB at pH 7.5. The column was eluted with a 400 mL linear gradient (0.1-0.9 M) TEAB. Fractions containing biotinyl-dUTP or biotinyl-UTP that eluted between 0.55 and 0.65 M TEAB were desalted by rotary evaporation in the presence of methanol and redissolved in water. Occasionally a slightly fuzzy solution was obtained. This turbidity caused by a contaminant in some TEAB solutions was removed by filtration through a 0.45 mm filter. For long-term storage, the nucleotides were converted to sodium salts by briefly stirring the solution in the presence of "Dowex" TM 50 (Na<+->form). After filtration, the nucleotide was precipitated by the addition of three volumes of cold ethanol, washed with ethyl ether, dried in vacuo over sodium hydroxide pellets and stored in a desiccator at -20°C. For immediate use, the nucleotide solution was made 20 mM in tris-HCl at pH 7.5 and adjusted to a final nucleotide concentration of 5 mM. Stock solutions were stored frozen at -20°C...

Elemental analysis of the bio-dUTP and bio-UTP products gave the following results.Bio-dUT<P>(<C>22<H>3Q<N>5<0>lg P3S1Na4• 1 H20). Theoretical: C, 29.80, H 3.38, N 7.89, P, 10.47,

S, 3.61. Found: C, 30.14 H, 3.22, N, 7.63, P, 10.31;

S, 3.70.Bio-UT<P>(C0~ H 3Q Nc 0, n P0 Sn Na/ 3 H„0) :

2 2 30 5 1 i) o 1 4 z Theoretical: C, 29.15, H, 3.19,. N, 7.45, P, 9.89, S, 3.41. Found; C, 28.76; H, 3.35, N, 7.68, P, 9.81, S, 3.32.

-The spectral properties of bio-dUTP and bio-UTP at pH 7.5

[X max, 289 nm (e= 7,100): X max, 240 nm (e = 10,700):

X min, 262 nm ( e= 4,300)] reflects the presence of an exo-cyclic double bond in conjugation with the pyrimidine ring. These nucleotides also give a strong positive reaction (an orange-red color) when treated with p-dimethylaminocinnaldehyde in ethanolic sulfuric acid, a method used for biotin determination (D.B. McCormick and J.A. Roth, Anal. Biochem. , _34, 326, 1970) . However, they no longer react with ninhydrin, a characteristic reaction for AA-dUTP and the AA-UTP starting materials.

Examples 3 and 4 Synthesis of biotinyl-CTP and biotinyl-dCTP

CTP and dCTP were a) mercuriated, b) reacted with allylamine and

c) biotinized with NHS-biotin, essentially as described in Example 1. CTP (56.3 mg, 0.1 mmol) or dCTP (59.1 mg,

0.1 mmol) was dissolved in 20 ml of 0.1 M sodium acetate buffer at pH 5.0 and mercuric acetate (0.159 g, 0.5 mmol) added. The solution was heated at 50°C for 4.5 hours and then cooled on ice. Lithium chloride (39.2 mg, 0.9 mmol) was added and the solution extracted 6 times with ethyl acetate. The nucleotide products in the aqueous layer were precipitated by the addition of three volumes called ethanol and the precipitate collected by centrifugation. The precipitate was washed with absolute ethanol, ethyl ether and then air dried. These products were used without further purification for the synthesis of resp. AA-CTP and AA-dCTP. The mercuriated nucleotides were dissolved in 0.1 M sodium acetate buffer at pH 5.0 and adjusted to a concentration of 10 mM (92 OD/ml at 275 nm). 0.6 ml (1.2 mmol) of a 2.0 M allylamine acetate stock solution (prepared as described in Example 1) was added to 10 ml of nucleotide solution (0.1 mmol) followed by the addition of I^PdCl^ (32, 6 mg, 0.1 mmol), dissolved in 1/6 ml of water. After standing at room temperature for 24 hours, the solution was filtered through a 0.45 mM membrane to remove metal precipitates. The filtrate was diluted five times and applied to one 50 ml column of DEAE-"Sephadex" A-25, which had been previously equilibrated with 50 mM TEAB at pH 7.5. The nucleotide products were fractionated by applying a

500 ml linear gradient (0.05-0.6 M) TEAB at pH 7.5. The desired product was in the largest UV-absorbing portion that eluted between 0.28 and 0.38 M salt. The combined samples

was desalted by rotary evaporation, dissolved in 0.5 M triethylammonium acetate at pH 4.2 and final purification achieved by HPLC chromatography on columns of Partisil 0DS-2, using 0.5 M triethylammonium acetate as eluent. Appropriate fractions were collected, lyophilized and the products dissolved in water. The nucleotides were converted to the Na<+> salt by brief stirring in the presence of "Dowex"TM 50 (Na<+> form). After filtration to remove the "Dowex" resin, the nucleotides were precipitated with the addition of 3 volumes of cold ethanol. The precipitate was washed with ether and then air dried. Analytical results: AA-dCTP (C12 H1?<N>4<0>13<P>3<N>a4'2H20), theory,

C, 22.29, H, 2.63, N, 8.67, P, 14.40. Found C, 22.16,

H 2.89, N, 8.77, P, 14.18. AA-CT<P>(<C>12H1?N4014Na4<*>2H20), theory C, -21.75, H, 2.57, N, 8.46, P, 14.01, Found, C, 22 .03, H, 2.47, N, 8.69, P, 13.81, Spectral properties in 0.1 M borate buffer at pH 8.0, X max 301 nm (e = 6,400)

X min 271'nm (e= 3,950)X max 250 nm (e = 9,700). Both AA-dCTP and AA-CTP give a positive ninhydrin test.

AA-CTP (6.6 mg, 0.01 mmol) or AA-dCTP (6.4 mg, 0.01 mmol) was dissolved in 5 mL of 0.1 M sodium borate buffer at pH 8.5,

and NHS-biotin (3.4 mg, 0.01 mmol) dissolved in 0.2 mL of dimethylformamide was added. After standing at room temperature

for 4 hours the sample was chromatographed on a 10 ml column of DEAE-"Sephadex" A-25 using 150 ml of linear gradient (0.1-0.9 M) TEAB at pH 7.5 as eluent. Fractions containing biotinyl-CTP or biotiny1-dCTP, which eluted between 0.50 and 0.60 M TEAB, were pooled, desalted by rotary evaporation, and after adjustment to a final concentration of 5 mM in 0.02 M Tris-HCl buffer at pH 7.5, frozen-at -20°C. Produktehe gives a strong positive reaction to biotin with p-dimethylaminocinnamaldehyde in ethanolic

••'sulphuric acid, but gives a negative test for primary amines when

they are sprayed with ninhydrin. Further structural characterization of these products is underway.

Examples 5 and 6

Synthesis of iminobiotinyl-UTP and iminobiotinyl-dUTP Iminobiotin hydrobromide was prepared from biotin as described previously (K. Hofmann, D.B. Melville and V. du Vigneaud,

J. Biol. Chem., 141, 207-211, 1941, K. Hofmann and A.E. Axelrod, Ibid., 187, 29-33, 1950). N-Hydroxysuccinimide (NHS) esters of iminobiotin were prepared using the procedure previously described for the synthesis of NHS-biotin (H. Heitzmann and F.M..Richards, Proe. Nat.Acad. Sei. USA, 7J.' 5537 , 1974). AA-UTP (7.0 mg, 0.01 mmol) or AA-dUTP (6.3 mg, 0.01 mmol) prepared as described in Example 1 (part b) was dissolved in 5 mL of 0.1 M sodium borate buffer at pH 8.5, and NHS-iminobiotin (3.5 mg, 0.01 mmol), dissolved in 0.5 ml of dimethylformamide, was added. The reaction mixture was allowed to stand at room temperature for 12 hours and then applied directly to a 10 ml column of DEAE-"Sephadex" A-25, which had previously been equilibrated with 0.05 M TEAB at pH 7.5. The column was eluted with a 150 mL linear gradient (0.05-0.6 M) TEAB. Fractions containing iminobiotin-UTP or iminobiotin-dUTP, which eluted between 0.35 and 0.40 MTEAB, were desalted by rotary evaporation in the presence of methanol and dissolved in water. The products contained a small amount of allylamine-nucleotide adduct as a contaminant, judged by a weak positive result in the ninhydrin test. Final purification was achieved by affinity chromatography on avidin-Sepharose. Fractions of the crude product, made 0.1 M with sodium borate buffer at pH 8.5, were applied to a 5 ml column of avidin-sepharose and washed with 25 ml of the same buffer. The column was then washed with 50 mM ammonium acetate buffer at pH 4.0, which eluted the desired iminobiotin nucleotide product in a sharp peak. The nucleotide was precipitated by adding 3 volumes of cold ethanol, washed with ethyl ether, dried in vacuum over sodium hydroxide pellets and stored in a desiccator at -20°C. The products were characterized by elemental analysis, as well as by spectral and chromatographic properties.

Examples 7 and 8

Synthesis of NAGE-UTP and NAGE-dUTP

Allyl (3-amino-2-hydroxy-)propyl ether, abbreviated NAGE,

was prepared from allyl glycidyl ether (Age) (obtained from Aldrich Chemical Co.). 10 ml of Age (84 mmol) was added slowly (in a fume hood) to 50 ml of 9 M ammonium hydroxide and the mixture was allowed to stand at room temperature for 6 hours. Excess ammonia was removed by rotary evaporation under reduced pressure to give a viscous yellow oil. Analysis of this product by proton NMR showed that it had the desired structure. 5-mercury-dUTP (0.1 mmol) or 5-mercury-UTP (0.2 mmol) was dissolved in 2-4 ml of 0.2 M sodium acetate buffer at pH 5.0, and a 16-fold molar excess of NAGE- adjusted to pH 5.0 with acetic acid before use,

was added. The final reaction volumes (4.3 and 8.4 ml) had nucleotide concentrations of 43 and 42 mM,

One equivalent of K 2 PdCl 4 (0.1 or 0.2 mmol) was added

to start the reaction. After standing at room temperature for 118 hours, the reaction mixture was filtered through 0.45 µM membranes, the samples diluted fivefold and chromatographed on columns of DEAE-"Sephadex" A-25, using linear gradients (0.1-0, 6 M) of sodium acetate. Factions that

contained the desired products, judged by their UV spectra and characteristic HPLC elution profiles on "Partisil" ODS-2, were collected, diluted and further purified by rechromatography on DEAE-"Sephadex" using narrow gradients (0.1-0 .5M of ammonium bicarbonate at pH 8.5. Under these conditions, the bulk of NAGE-dUTP (or NAGE-UTP) could be clearly separated from residual contaminants. Proton NMR spectra were obtained at this step of the purification after the nucleotides were lyophilized and redissolved in D 2 O . For elemental analysis, the products were converted to their sodium salt form. Typical "analytical results: Nage-dUT<P>(<C>15H22<N>3<0>l<g>P3Na42 H20), theory, C, 24.99 , H, 3.63, N, 5.83, P, 12.88.

Found, C, 25.39, H, 3.71, N, 5.63, P 12.88.

Example 9

Use of tagged DNA sequences

I. Karyotyping

(a) From 100 to 200 clones are selected from a human gene pool. They are labeled as described above, and for each clone their hybridization site or sites were located visually or with a low-light video system. For those clones that correspond to a gene from a unique sequence, this determines the location of the cloned DNA on a particular human chromosome. Several clones are obtained for each chromosome. Each of these labeled clones can be used to identify particular chromosomes. They can also be used in combination to identify each of the 46 chromosomes as one of the 22 autosomal pairs or X or Y. By allowing one set of labeled clones to hybridize to the chromosomes and then adding a fluorescent dye to the label, the set of clones and their location can be made visible and will fluoresce with a special color. A second set of labeled clones could then be used and reacted with a second fluorescent dye. The same procedure can be repeated a number of times. One can then, if desired, have several sets of fluorescent "labels" attached to them

.the cellular DNA at different but specific locations on each of the chromosomes. These markers can be used for visual or computer-controlled automatic karyotyping. (b) For automatic karyotyping, one set of clones can be used to identify the approximate location of each of the 4 6 chromosomes by finding sets of spots corresponding to the number of labeled sites on each chromosome.

It is thus possible by computer analysis of the digitized "images" to determine whether the chromosomes are suitably spread out for further analysis. If they are suitably spread out, computer analysis can be used to identify each of the individual chromosomes by locating and distributing the marked spots on each of them.

Using the fact that the fluorescent spots can be placed on special localizations on each chromosome,

either manual or automatic karyotyping can be performed much more efficiently than without such labels.

II. Diagnosis of genetic diseases

By selecting those clones that bind specifically to a particular chromosome, such as e.g. No. 23, it is possible to count the number of copies of the particular chromosome in a cell even if the chromosome is not condensed at metaphase. Thus, when fetal cells are admitted for prenatal diagnosis of trisomy 21,

the diagnosis can be made even if the chromosomes are not condensed at metaphase. If necessary, two sets of tags can be used, one that would be specific for chromosome 23 and one for another chromosome. By measuring in each cell the ratio between the two labels, which may be of different colors, it is possible to identify the cells showing an abnormal number of chromosome number 23. This method can be used either on slides with a low-light video system or a flow cytometer system using laser excitation. It can be used to determine any abnormal chromosome numbers.

III. Microorganism detection and identification

Labeling of specific sequences of DNA as described above enables the identification and counting of individual bacteria. In order to identify the individual bacteria to which a particular fragment of DNA hybridizes, the sensitivity must be such that a single labeled structure can be detected. This can be done by using a low-light video system and computer summarization of images or by using another

.^device for amplifying the slide. A flow system can also be used if the sensitivity can be made sufficiently large. If the bacteria are immobilized on

a slide, their location can be found and the number of such fluorescent spots counted. This will give a number of all the bacteria that contain DNA that can hybridize

sere with the specific clone used. If the clone is chosen to be specific for a particular strain or bacterium, then the number of organisms of this strain can be counted. In addition, any antibiotic resistance for which a particular gene has been identified can be similarly characterized by using as a probe the DNA sequence contained in the antibiotic resistance gene. In addition, a probe can be used which is specific for a resistance plasmid, containing one or more antibiotic resistance genes.

In addition to individual bacteria, groups of bacterial cells of a particular strain can be detected and their number determined if they are in a small spot, so that the total fluorescence specific to the hybridized DNA in the spot can be measured. In this way, the number of organisms containing a specific DNA sequence can be measured in a mixture of bacteria.

As further background regarding the use of the biotin polynucleotides indicated above, the article by Langer et al in Proe. Nati. Acad. Pollock. USA

and the publication of P.R. Langer-Safer, M Levine and D.C. Ward

in Genetics entitled "An Immunological Method for Mapping Genes on Drosophila Polytene Chromosomes", described method where biotinylated nucleotides are used as a probe for the localization of DNA sequences which are hybridized in situ

to Drosophila polytene chromosomes. In this application, these probes are detected by using affinity-purified rabbit antibiotin antibody as the primary antibody and fluorescent goat antirabbit antibody as the secondary antibody.

What is described in this publication by Langer-Safer et al in Genetics is also included in and made part of this description.

Other techniques using biotin-labeled reagents with avidin or enzyme-labeled avidin reagents are known for the detection and determination of ligands in a liquid medium,

see U.S. patent 4,228,237. It is also known to carry out

gene enrichments based on. avidin-biotin reaction, especially when applied to Drosophila ribosomal RNA genes, see the publication of J. Manning, M. Pellegrini and N. Davidson in Biochemistry, vol. 16, no.<7>, pages 1364-1369 (1977). Other publications of background interest in the practice of the present invention are the article by D.J. Eckermann and R.H. Symons entitled "Sequence at the Site of Attachment of an Affinity-Label Derivative of Puromycin on 23-S Ribosomal RNA of Escherichia coli Ribosomes", J. Biochem, 82, 225-234 (1978), the article by S.B. Zimmerman, S,R. Kornberg and A. Kornberg entitled "Glucosylation of Deoxyribonucleic Acid-II -Glucosyl Transferases from T2- and T6-Infected Escherichia coli in The Journal of Biological Chemistry, vol. 237, no. 2, February 1962, and the article by J. Josse and A. Kornberg "III. a- and 3-Glucosyl Transferases from T4-Infected Escherichia coli", which also appears in The Journal of Biological Chemistry, vol. 2 37, no. 6, June 1962.

Of further interest in connection with the practice of the present invention are the publications appearing in J. Biol. Chem., Vol. 236, No. 5, May 1961, pages 1487-1493, the same publication vol. 237, No. 4, pages 1251-1259 (1962), the same publication vol. 239, No. 9, pp. 2957-2963 (1964). Of particular interest is the article appearing in The Journal of Histochemistry and Cytochemistry, vol. 27, No. 8, pages 1131-1139 (1979) and in the publication Nucleic Acids Research, vol. 5, No. 9, 1977, pages 2961-2973. Also of interest is the article appearing in the publication Biochimica et Biophysica Acta by A. De Waard entitled "Specificity Difference Between the Hyroxymethylcytosine 3-Glucosyl-Transferases Induced<x>by Bacteriophages T2, T 4 and T6", pages 286-304 ., and also the article by T.W. North and C.K. Mathews with. entitled "T4Phage-Coded Deoxycytidylate Hydroxymethylase: Purification and Studies in Intermolecular Interactions", published by Academic Press, 1977, pages 898-904 and the article by E.A. Bayer and M. Wilchek entitled "The Use of Avidin-Biotin Complex as a Tool in Molecular Biology in Methods of Biochemical Analysis, vol. 26, pages 1-45 (1980).

Other techniques useful in the practice of the present invention include cut transfer of DNA using DNA polymerase. A technique for carrying out section transfer is described in the article by P.W. Rigby, M. Dieckmann, C. Rhodes and P. Berg, entitled "Labeling Deoxyribonucleic Acid to High Specific Activity in vitro by Nick Translation with DNA Polymerase" in J. Mol. Biol. (1977), 113, 237-251. When it comes to the recovery of streptavidin, which e.g. from a culture fluid of Streptomyc. es avidinii, describes the article by K. Hofmann, S.W. Wood, C.C. Brinton, J.A. Montibelle and

F.M. Find titled "Iminobiotin Affinity Columns and their Application to Retrieval of Streptavidin" in Proe. Nati. Acad. Pollock. United States, vol. 77, No. 8, pages 4666-4668 (1980),

a suitable method for recovering streptavidin from a streptavidin-containing material, such as e.g. from a culture fluid. Streptavidin is useful as a reagent in one of the embodiments of the invention.

The aforementioned publications are hereby introduced and made into

part of this description.

According to the implementation of this invention, nucleotides are modified preparatively, such as e.g. at the 5-position of pyrimidine or the 7-position of purine, for the production from there of nucleotide probes which are suitable for attachment to or mixing into DNA or other nucleic acid material. In the practice of the present invention, nucleotides, i.e. nucleic acid, are preferably modified in a non-cleaving manner so that the resulting modified nucleotides can be incorporated into nucleic acids and once incorporated into the nucleic acids, the modified nucleotides do not significantly affect the formation or stabilization of the double helix formed from the resulting nucleic acids containing the modified nucleotides. The non-cleavable modification of the nucleotides and nucleic acids and those nucleic acids containing such modified nucleotides contrasts with those modifications of nucleotides which are characterized as cleavage modifications in the sense that the resulting cleavage-modified nucleotides and nucleic acids containing the same block proper double- spiral formation. When practicing the present invention, the nucleotides are desirably modified in the 5-position of the pyrimidine or the 7-position of the purine. The nucleotides thus modified are non-cleavable modified and nucleic acids containing such nucleotides are capable of forming a double helical arrangement.

In another aspect of the practice of the present invention, various methods are applicable for labeling DNA in a non-cleaving manner. Biotin is added, for example, to the end of a DNA or RNA molecule. The addition of biotin is carried out by the addition of a ribonucleotide. The 3',4<1->vicinal hydroxyl groups are oxidized by periodate oxidation and then reduced by borohydride in the presence of biotin hydrazide. Alternatively, carbodiimide can also be used to link biotin to the aldehyde group.

Another technique for labeling nucleic acid material such as DNA or RNA involves adding a large marker to the end of a DNA or RNA molecule. One example of this technique is the addition of a molecule, e.g. lysylglycine, where the amino groups are labeled with biotin.

Another example would be to follow the method indicated above, but use carbodiimide as a cross-linking agent. A further example of this technique would be to prepare a biotinylated dA:dU double helix polymer and bind this polymer to the probe prepared according to the present invention.

Another technique for labeling DNA in a non-cleaving manner involves the isolation of dPyrTP with a putricin or spermidine at the 5-position from PS16 or phage-infected cells. If desired, dPyrTP is made from phage DNA and dPyrTP is phosphorylated followed by modification of the polyamine side chain using the standard nucleophilic reagent NHS-biotin.

Another technique for labeling DNA in a non-cleaving manner involves the addition of glucose to 5-hydroxymethylcytosine (5 HMC) in DNA using T4 phage glycosylating enzymes followed by sieving using a lecithin-based assay.

Yet another method for labeling DNA in a non-cleaving manner involves 5-HMC triphosphate prepared from the hydrolysis of T4 DNA followed by phosphorylation of 5HMCMP to 5HMCTP.

5HMCTP is then incorporated into DNA using polymerase I. Thus, any DNA can be modified so that 5 HMC is introduced in a non-cleaving manner.

A method for labeling DNA in a weakly cleaving manner comprises reacting nucleic acids in double helix form with allylating reagents such as e.g. benz(o)pyrene-diol-epoxide or aflatoxin. Under appropriate conditions, the N 2 group is alkylated in guanine, the N 4 group in adenosine or the N 4 group in cytosine. These modified nucleotides can be directly detected with antibodies or can be used as binding arms for the addition of a label molecule such as e.g. biotin.

The following examples illustrate different embodiments when implementing the present invention:

- Example I

Biotinyl N-hydroxysuccinimide ester (BNHS) was prepared according to the method of Bécker et al, P.N.A.S. 68 26o4

(1971). Biotin (0.24 g, 1.0 mmol) was dissolved in 5 ml of dry dimethylformamide. Dicyclohexylcarbodiimide (0.21 g, 1.0 mmol) and N-hydroxysuccinimide (0.12 g, 1.0 mmol) were added and the solution stirred at room temperature for 15 h. After filtering the resulting precipitate, the filtrate was evaporated under reduced pressure and the residue washed twice with ethanol and recovered from hot isopropyl alcohol to give a white crystalline product with a m.p. at 216-218°C.

Example II

Biotinyl-1,6-diaminohexane-amide was prepared as follows:

A solution of 1>6-diaminohexane (320 mg, 2.0 mmol), dissoln

in 50 ml of water, was brought to pH 8.5 by the addition of carbon dioxide. Biotinyl N-hydroxysuccinimide ester (100 mg, 0.29 mmol), dissolved in 10 mL of dimethylformamide, was added. After 18 hours at room temperature, the mixture was evaporated and the residue washed with ether and then dried in a desiccator.

Example III

Polybiotinylated poly-L-lysine was prepared using the following procedure. Polylysine (100 µmol lysine) dissolved in 2 mL of 0.1 M sodium borate, pH 8.5 was added to biotinyl N-hydroxysuccinic acid imide ester (17.5 mg, 50 µmol) dissolved in 0.5 mL dimethylformamide. After stirring at room temperature for 18 hours, the mixture was dialyzed against 10 mM Tris buffer, pH 7.5.

Example IV

Oligodeoxyribonucleotide was end-labeled using cytidine-5'-triphosphate and terminal transferase as follows. Purified Phage DNA, alkalized with 0.2 N sodium hydroxide and diluted to 2 & 26Q enethers/ml in potassium cacodylate (0.1M), tris-base (25 mM), cobalt chloride (1mM) and dithiothreitol (0.2M) were used To this DNA solution (1ml) were added cytidine-5'-diphosphate (10 mmol) and terminal transferase (200 units). After incubation at 37°C for 5 to 8 hours, the reaction was stopped by the addition of neutralized phenol (100 µl), 0.5M EDTA (100 µl) and 1% sodium dodecyl sulfate (100 µl). DNA was purified by gel filtration chromatography through "Sephadex" G-100 followed by precipitation with ethanol.

Example V

Biotin and polybiotinylated poly-L-lysine were linked to oligoribonucleotides using a carbodiimide coupling method described by Halloran and Parker, J. Immunol., 96 373 (1966). As an example, DNA (1 µg/ml), 1 ml)

in tris buffer pH 8.2 treated with 0.1 N sodium hydroxide denatured by boiling for 10 minutes and rapid cooling in an ice bath. Biotinyl-1,6-diaminohexanamide (2 mg, 6 μmol) or polybiotinylated poly-L-lysine (2 mg) and 1-ethyl-3-diisopropylaminocarbimide-HCl (10 mg, 64 μmol) were added, and pH adjusted back to 8.2. After 24 hours at room temperature in the dark, the mixture was dialyzed against 10 mM Tris-buffered saline. DNA was precipitated with ethanol.

Example VI

Biotin, conjugated to cytochrome C, was prepared by the following procedure. To a solution of cytochrome C (10 mg) in 1 ml of 0.1 M sodium borate, pH 8.5, was added biotinyl N-hydroxysuccinimide ester (10 mg, 29 μmol)

in 1 ml of dimethylformamide. After 4 hours at room temperature, the biotinylated protein was purified by gel filtration chromatography through a Sephadex G-50 column.

Example VII

Formaldehyde coupling of cytochrome-C-biotin and polybiotinylated poly-L-lysine to oligodeoxyribonucleotides was performed using a method similar to that described by Manning et al, Chromosoma, 53, 107 (1975). Oligodeoxyribonucleotide fragments obtained by sodium hydroxide treatment of purified DNA (100 µg/ml in 10 mM triethanolamine, pH 7.8) were denatured by boiling for 10 min followed by rapid cooling in ice. Cytochrome-C-biotin 0.05 g ml or polybiotinylated poly-L-lysine solution (0.05 ml) dissolved 3 mg/ml in 10 mM triethanolamine, pH 7.8 was added to 1 ml of the denatured oligodeoxyribonucleotide solution together with 0.1 ml of 6% formaldehyde in 10 mM triethylanolamine , pH 7.8. After stirring at 40°C for 30 minutes, the mixture was dialyzed against the same buffer. The oligodeoxyribonucleotide-biotin complex was finally purified by gl-filtration chromatography on "Sephadex" G-100 followed by precipitation from ethanol.

Example VIII

Double-stranded polydeoxyadenylic acid:polybiotinylated deoxyridylic acid was synthesized as follows. The double-stranded oligonucleotide polydeoxyadenylic acid:polythymidyl acid (20 µg) with a length of 300 base pairs, dissolved in 200 µl exonuclease III buffer consisting of tris-HCl pH 8.0 (70 mM), magnesium chloride (1.0 mM) and dithiothreitol (10 mM) was incubated with 100 units of exonuclease III for 20 min at 20°C. The partially digested oligonucleotide was immediately extracted with phenol, and the DNA was precipitated with 70% aqueous ethanol. The partially digested oligonucleotide was redissolved in 20 µl of 5 mM tris-HCl pH 7.6 and incubated at 20 oCi for 2 hours in a reaction mixture containing 2'-deoxy-adenosine-5'-triphosphate (15 µM), thymidine-5' -triphosphate (the amount determines the degree of substitution) and biotinylated 5-(3-amino-1-propene) 2'-deoxyuridine-5<1->triphosphate (5 µM), Klenow DNA polymerase I (200 units) dissolved in 0, 1 mM potassium phosphate, pH 8.0 at a concentration of 0.2 units/µl. The biotinylated poly-dA:poly-dT, biotinyl dU was purified by gel filtration chromatography on Sephadex G-100. DNA

was precipitated with ethanol and redissolved in 20 µl solution containing sodium acetate pH 4.6 (30 mM), sodium chloride (50 mM), zinc sulfate (1 mM) and glycerol (5%). S^-nuclease (200 units) was added and the reaction mixture was incubated at 37°C

for 10 minutes. The reaction was quenched with 1 ml of ammonium acetate (4 M) and 6 ml of ethanol. DNA was purified again by G-100 gel filtration chromatography and ethanol precipitation.

Example IX

Linking of poly-dA:poly-dT, biotinyl-dU with oligo-deoxy-ribonucleotides was carried out as follows:

DNA fragments from alkali-treated, purified DNA (eg

described in Example VIII) was digested with S^-nuclease and repurified by phenol extraction and ethanol precipitation. Blunt-ended DNA fragments (1 µg) and poly-dA:poly-dT, biotinyl-dU (2 µg) were dissolved in 6 µl of a buffer containing tris-HCl pH 7.4 (66 mM), magnesium chloride (6.6 mM), adenosine triphosphate (24 mM) and dithiothreitol (1.0 mM). T4DNA ligase (50 units) was added and the volume adjusted

to 20^ul with water. The reaction mixture was incubated for 3

hours at 37°C. DNA was purified by gel filtration chromatography through "Sephadex" G-100 and was precipitated with ethanol.

Example X

5-Hydroxymethyl-2<1->deoxycytidylic acid was prepared by enzymatic hydrolysis of non-glycosylated phage T4DNA.

Purified phage DNA (2mg), dissolved in 1 ml of 50 mM tris pH 7.4 and

10 mM magnesium chloride was incubated for 20 hours with deoxyribonuclease I at 37°C. The pH was adjusted to 9.0 and sodium chloride (20 mM) added. •Snake venom phosphodiesterase (0.05 g units in 0.5 ml water) was added and the incubation continued at 37°C for 5 hours. An additional 0.05 units of phosphodiesterase was added and the incubation continued in

•18 hours. Nucleotides were separated by gel filtration chromatography through Sephadex G-50: 5-hydroxymethyl-2'-deoxycytidylic acid was purified by high-pressure liquid chromatography

in the opposite phase.

Example XI

5-(4-aminobutylaminomethyl)-2'-deoxyuridylic acid was obtained by enzymatic hydrolysis of DNA from subject ØW-14. The phage was grown on Pseudomonas acidovorans 29 according to Kropinski and Warren, Gen. Virol. 6, 85 (1970) and phage DNA purified

according to Kropinski et al, Biochem. 12, 151 (1973). DNA was enzymatically hydrolyzed with deoxyribonuclease I and

snake venom phosphodiesterase using the procedure described above (Example X). 5-(4-aminobutylamino-methyl)-2'-deoxyuridylic acid was purified by reverse phase high pressure liquid chromatography.

Example XII

Biotinylated 5-(4-aminobutylaminomethyl)-2<1->deoxy-uridylic acid was prepared as follows: biotinyl-n-hydroxysuccinimide ester (70 mg, 0.2 mmol) dissolved in 1 mL of dimethylformamide was added to 5- (4-aminobutylaminomethyl)-2'-deoxyuridylic acid in 20 ml of 0.1 M sodium borate pH 8.5. After 4 hours, the solution was concentrated to 0.5 ml by evaporation,

and the biotinylated nucleotide was purified by reverse phase high pressure liquid chromatography.

Example XIII

5-fprmyl-2'-deoxyuridine prepared according to Mertes and Shipchandler, J. Heterocyclic Chem. 1, 751 (1970). 5-Hydroxymethyluricil (1 mmol) dissolved in 20 ml of dimethyl sulfonate was heated at 100°C with manganese dioxide (2.5 mmol) for 15 minutes. The solvent was evaporated under reduced pressure. The residue was taken up in hot ethanol and recrystallized from ethanol to give 5-formyluracil, 5-formyluracil (0.10 g) was silylated and dissolved in dry acetonitrile (2.5 mL), 2-deoxy-3,5-di- O-p-tolyl-D-ribofuranosyl chloride (Bhat, Syn. Proe, in Nucleic Acid Chem., vol. I, page 521 (1968) (0.22 g) and molecular sieves (0.2 g) were added and the mixture was stirred at 25°C for 40 hours under anhydrous conditions. The mixture was filtered and evaporated. The resulting oil was treated with anhydrous ethanol (2 mol) and chromatographed on silica gel to obtain the partially pure anomer which was recrystallized from ethanol (m.p. . 195-196°C). The toluyl groups were removed by reaction between the product in methanolbenzene and sodium methoxide. The mixture was neutralized with "Dowex" 50 (H+). 5-formyl-2'-deoxyuridine was recrystallized from ethanol (m.p. .175-176°C).

Example XIV

Biotin was coupled to 5-formyl-2<1->deoxyuridine as follows: To 5-formyl-2'-deoxyuridine (0.320 g , 1.0 mmol) dissolved

in 300 ml of 0.05 M sodium borate, biotinyl-1,6-diaminohexanamide (0.74 g, 2 mmol) was added. After stirring for 1 hour, sodium borohydride (0.2 g, 5 mmol) was added and stirring continued for an additional 4 hours followed by the addition of 8 mL of 1M formic acid. The biotinylated compound was purified by reverse phase HPLC elution with methanol:0.5 M triethylammonium acetate, pH-4.0.

Example XV

Biotin was coupled to 5-amino-2<1->deoxyuridine as follows: 5-amino-2<1->deoxyuridine (0.24 g, 1 mmol), biotin (.0.25 g, 1 mmol) and dicyclohexyl carboimide (0.21 g, 1 mmol) was dissolved in dry dimethylformamide and stirred at room temperature overnight. After filtration and evaporation of the solvent, the residue was washed with ether. The biotin-linked product was purified by reverse phase high pressure liquid chromatography using a water-methanol gradient.

Example XVI

5-(oxy)acetic acid-2<1->deoxyuridine was prepared according to the method of Deschamps and DeClerq, J. Med. Chem., 21 228 (1978). 5-Hydroxy-2-deoxyuridine (282 mg, 1.15 mmol) was dissolved in 1.16 mL, 1N potassium hydroxide (1.16 mmol) followed by iodoacetic acid (603 mg, 3.4 mmol) in 1 mL of water added. After reaction at room temperature for 48 hours, 1N HCl (1.06 mL) was added. Concentration of this solution

and addition of ethanol gave a precipitate which was filtered, washed with cold ethanol and recrystallized from hot ethanol.

Example XVII

Biotinyl-1,6-diaminohexane-amide was coupled to 5-(oxy)acetic acid-2<1->deoxyuridine as follows: Biotinyl-1,5-diaminohexane-amide (0.74 g, 02, mmol), 5- (oxy)acetic acid-2<1->deoxyuridine (0.60 g, 02, mmol) and dicyclohexyl carboimide

0.41 g, 0.2 mmol) was dissolved in 5 ml of dry dimethylformamide and allowed to stand overnight at room temperature. The reaction mixture was then filtered and the solvent removed by evaporation. The residue was washed with 0.1 N HCl and ether. The biotinylated uridine derivative was purified by reverse phase high pressure liquid chromatography using a water-methanol gradient.

Example XVIII Phosphorylation of 5-substituted pyrimidine nucleosides was carried out using the general procedure described below for biotinylated 5-(oxy)acetic acid-2'-deoxyuridine. The nucleotide (0.16 g, 0.5 mmol) was dried by repeated evaporation from dry pyridine and redissolved in 10 ml of dry pyridine. Monomethoxytrityl chloride (0.3 g, 0.8 mmol) was added and the mixture stirred at room temperature in the dark for 18 h. The solution was diluted with chloroform (200 mL) and extracted with 0.1 M sodium bicarbonate. The organic layer was dried and evaporated. The tritylated nucleoside was redissolved in dry pyridine (20 mL) and acetylated by reaction at room temperature with acetic anhydride (0.1 mL, 20 mmol). The mixture was cooled to 4°C and methanol (40 mL) added. After stirring for 10

hours at room temperature, the reaction mixture was concentrated by evaporation. The compound was detritylated by dissolving in 1% benzenesulfonic acid in chloroform (20ml). After evaporation of the solvent, the nucleoside was purified by chromatography on silica gel and eluted with 2% methanol:chloroform. The 3'-acetylated nucleoside was dried by repeated evaporation of dry pyridine. A mixture of phosphorus oxychloride (100 µl, 1 mmol)', (1-H), 1,2,4-triazoic (140 mg, 2.2 mmol) and triethylamine (260 µl, 2.0 mmol) was stirred in 5 ml of water-liter dioxane at 10-15°C for 30 minutes and at room temperature for 1 hour. This was added to 3'-acetylated nucleoside,

and the mixture stirred at room temperature for 1 hour after which it was cooled to 0°C. Water (5 mL) was added and the reaction mixture was stirred at room temperature for 18 h.

Barium chloride (100 mg., 5 mmol) was added and the barium salt of the nucleotide collected by filtration. The salt was washed with water and ether. The barium salt was converted to the sodium salt by stirring with "Dowex"50 (Na<+> form) in 10 ml of water for 4 hours at room temperature. 2N sodium hydroxide (2N, 10ml) was added and the reaction stirred for 15 minutes at room temperature, after which it was neutralized by addition of excess "Dowex" 50 (H<+>) form. The deacetylated nucleotide was concentrated by evaporation and purified by reverse phase high pressure chromatography.

Example XIX

5-substituted pyrimidine triphosphates were chemically produced

prepared from their respective 5<1->monophosphates using the method of Michelson, Biochem Biophys Acta, 91, 1,

(1964). As an example, 5-hydroxymethyl-2<1->deoxycytidine-5'-triphosphate should be given. The others were produced in a similar way. 5-Hydroxymethyl-2<1->deoxycytidylic acid (free acid) (0.63 g, 0.2 mmol) was converted to its tri-n-octylammonium salt by suspension in methanol and addition of tri-n-octylammonium hydroxide (0.74 g, 0.2 mmol). The suspension was refluxed until a clear solution was obtained and the solvent removed under vacuum.

The salt was dried by dissolution in and subsequent evaporation from dry pyridine several times. For the salt, dissolved in dry dimethylformamide (0.1 ml) and dioxane (1 ml) diphenylphosphochloridate (0.1 ml) and tri-n-butylamine (0.2 ml) were added. After 25 hours at room temperature, the solvent was removed and ether was added to precipitate the nucleoside 5'-diphenyl pyrophosphate. This was dissolved in dioxane (0.5 ml)" and a solution of di(tri-n-butylammonium) pyrophosphate (0.5 mmol) in 1 ml pyridine was added. After •-45 minutes at room temperature, the mixture was concentrated under vacuum to a small volume. The crude product was precipitated with ether. This was dissolved in 0.1 M phosphate buffer pH 8.0. The triphosphate was purified by chromatography on DEAE cellulose and eluted with a gradient of 0.1 to 0.6 M triethylammonium-

bicarbonate pH 7.5.

Example XX

DNA was labeled with 5-substituted pyrimidine triphosphates by section transfer of DNA in the presence of the appropriate triphosphate. An example follows of labeling purified DNA with biotinylated 5-formyl-2<1->deoxyuridine. DNA (20 µg/ml) was incubated at 14°C in the presence of magnesium chloride (5 mM) 2'-deoxycytidine-5<1->triphosphate (15 mM), 2<1->deoxyadenosine-'5 1-triphosphate (15 µM), 2'-deoxyguanosine-5'-triphosphate (15 µM), biotinylated 5-formyl-2'-deoxyuridine-5 1 -tri-phosphate (20 µM), activated pancreatic deoxyribonuclease I (13 mg/ml), E. coli deoxyribonuclease acid, polymerase I (40 units/ml) and tris-HCl, pH 7.4 (50 mM). After 2 h, the reaction was stopped by addition of 0.3 M EDTA (0.05 mL) followed by heating at 65°C for 5 min. Labeled oligonucleotide was purified by gel filtration chromatography through Sephadex. G-100 and precipitation from cold ethanol.

Example XXI

Precipitation of glucosylated DNA with Concanavalin A Reaction mixtures (1.0 ml) were prepared in 1.5 ml eppendorf tubes as follows:

"Reactions were initiated by the addition of concanavalin A (Con A). The solutions were mixed and allowed to stand at room temperature for 60 minutes. The tubes were centrifuged at 1200 g for 15-20 minutes. The supernatants were diluted and A260 was measured.

Since Con A absorbs at 260 nanometers, control solutions were prepared that lacked DNA but contained Con A. The absorbance for Con A was subtracted from the absorbance obtained in the complete reaction mixtures.

The results of this reaction are shown in the accompanying Figure 1.

Example XXII

Binding of glucosylated DNA to concanavalin A

3

Phage T4 DNA and phage DNA were labeled by introducing H-deoxyadenosine triphosphate into the DNA by section transfer according to the method of Rigby et al. T4 DNA was averaged to a specific activity of 5x10<5>cpm/microgram and an average double-stranded size of 5 kilobases. Lambda DNA was averaged to a specific activity of 3x10~<*>cpm/microgram and an average double-stranded size of 6.0 kilobases as determined by agarose gel electrophoresis. Unincorporated nucleotides were removed from the reaction mixtures by Bio-Gel P-60 chromatography.

Con-A Sepharose was prepared as described by the manufacturer (Pharmacia). 1 ml of deposited gel contained 18 mg bound

Con A. 1 ml columns were prepared in sterile Pasteur pipettes and were equilibrated with PBS (0.15 M NaCl,

0.01 M sodium-potassium^phosphate, pH 6.5).

H 3-DNA samples were prepared in 0.5 ml buffer (as described

in Example XXI, but without Con A). T4 DNA solutions contained 176,000 cpm/0.5 ml and DNA solutions contained 108,000 cp./0.5 ml. A 0.5 ml sample was applied to the column.

A 10.5 ml volume of buffer was passed through the column and the eluate fractions (0.33 m) were collected and poured into a Beckman LSC-100 spark counter in a 3.5 ml "reafluor cocktail" (Beckman). The results (Fig. 2A and 2B) show that non-glucosylated DNA was not bound, while glucosylated T4 DNA was bound to the column. The bound T4 DNA was removed by washing the column with a high pH buffer (Tris-HCl, pH 7.2-8.2).

Furthermore, consistent with the reaction between glucose

and Con A, mannose, when mixed into the buffer where DNA is applied to the column, prevents binding of glucosylated DNA to Con A-sepharose. Mannose-containing buffer (PBS containing 0.056 M mannose) also removes bound T4 DNA from Con A Sepharose (Figures 3A and 3B).

For further illustration of the implementation of the present invention directed at non-radioactive methods or techniques for samples of specific nucleic acids, the following example deals with the use of the sugar-lectin system. This example deals with the use of DNA as

by nature is not glycosylated, but instead a maltotriose group is added by section transfer as described here. The maltotriose-modified dUTP and DNA modified thereby bind specifically to a column of concanavalin A covalently bound to sepharose. With this technique and according to the implementation of the present invention, a method for specific labeling of nucleic acids with sugar has been achieved. As previously indicated, section transfer is only one of a number of techniques and methods possible for the production/tilting of the modified nucleic acids of the present invention.

Example XXIII

Lambda DNA was sectioned as described herein with maltotriose linked to 5-(3-amino-1-propenyl)-2'-deoxyuridine-5<1->triphosphate and<3>H-2'-deoxyadenosine-5< 1->triphosphate. Under these conditions, the DNA was substituted to 40% of its thymidine residues with maltotriose nucleotides and had a specific activity of 8x10^ counts per minute (cpm) per micrograms of DNA. A control sample of DNA substituted only with<3>H-dATP had a specific activity of 6x10"* cpm per microgram of DNA. The aliquoted DNA samples were purified free of reaction mixture constituents by Biogel P-60 chromatography as described herein.

The purified samples were applied to Con A-sepharose columns

as described in Figures 2A and 2B. The maltotriose-

labeled DNA was retained on the column when washed with PBS, but was removed by subsequent elution with 10M tris-HCl, pH 8.2 (Figure 4A). The unsubstituted tritiated DNA was not bound to the column at pH 7.4

(Figure 4B).

Example XXIV

Potentially immunogenic heptenes can be introduced in the 5-position

in uridine using a number of methods in the literature. 5-(perfluorobutyl)-2'-deoxyuridine was synthesized using the method of Cech et al, Nucl. Acids Res. 2, 2183 (1979). Copper-bronze was produced by reacting copper sulphite

(5 g, 20 mmol) with zinc powder (2 g) in 20 ml of water. The mixture was decanted, and the residue washed with water and then with 5% hydrochloric acid and water. Just before use, the solid (2 g) was activated with 2% iodine in acetone (20 ml). After filtration, the residue was washed with acetone:concentrated hydrochloric acid and so

with pure acetone. Activated copper-bronze (130 mg.2 mmol)

and 1-iodo-1<1>,2,2',3,3<1>,4,4'-heptafluorobutane (1.3 mg, 4 mol)

was stirred in 3 ml of dimethylsulfoxide at 110°C for 1 hour.

After cooling and filtering, 2<1->deoxyuridine (245 mg

1 mmol) added, and the mixture heated at 110°C for 1 hour. Water (5 mL) was added and the mixture extracted with ether. The ether extracts were dried and evaporated under reduced pressure

Print. The residue was chromatographed on a silica gel column and eluted with ethyl acetate.

Example XXV

Tubericidine was substituted in the 5-position by derivatizing the 5-cyano compound, toyokamycin. An example is the synthesis of 4-amino5(tetrazol)-5-yl)-7-(B-g-ribofuranosyl)pyrrolo[2,3-d]pyrimidine using the method of Schram and Townsend, J . Carbohydra. te , Nucleosides :Nucleotides 1, 38

(1974). Toyokamycin (1.0 g) dissolved in water (100 ml) and

glacial acetic acid (13 mL) was heated to reflux. Sodium azide (7.5 g) was added in 1.25 g portions over 10 hours. The solution was cooled to 5°C and the precipitated product collected, m.p. 276-277°C.

Example XXVI

5-cyano-2<1->deoxyuridine was prepared according to Bleckley et al, Nucl. Acis Res. 2, 683 (1975). 5-iodo-2<1->deoxyuridine

(1.0 g, 2.82 mmol) was dissolved in refluxing hexamethyldisilizane (HMDS) (10 mL). Excess HMDS was removed under reduced pressure and the resulting oil was dissolved in dry pyridine (50 mL). Cuprous cyanide (350 mg, 3.8 mmol) was added and the solution heated at 160°C for 20 hours. Pyridine was removed under reduced pressure and the residue extracted with toluene which was then evaporated. The residue was heated in 50% aqueous ethanol at 100°C for 2 hours. The pdohydrate was purified by reverse phase high pressure liquid chromatography and recrystallized from ethanol, m.p. 161°C.

Example XXVIl 4-Amino-5-amino-methylene-7-(O-D-2-deoxy-furanosyl)-pyrrolo[2,3-d]pyrimidine dihydrochloride was obtained as follows. 4-amino-5-cyano-7-(3~D-2-deoxy-furanosyl)pyrrolo[2,3-d]pyrimidine (Toyocamycin) (0.2 g)

was dissolved in hydrochloric acid (10 ml). 10% palladium-on-charcoal

(0.1 g) was added and the mixture hydrogenated at 2.8 kg/cm 2 for 5 hours at room temperature. After filtration, the water was evaporated at reduced pressure. The residue was triturated with ethanol and the product recrystallized from 50% ethanol.

Example XXVIII

5-amino-2'-deoxyuridine was prepared from 5-bromo-2'-deoxyuridine according to the method of Roberts and Visser,

J. Am. Chem. Soc. 14:665-669 (1952). 5-bromo-2<1->deoxyuridine

(2g, 6.2mmol) dissolved in liquid ammonia (20ml) was measured into a glass tube and heated at 50°C for 5 days. The tube was opened and the ammonia evaporated. 5-amino-2'-deoxyuridine was recrystallized from 5 ml of water and 75 ml of hot isopropyl alcohol.

Example XXIX

5-(Methylamino)-2<1->deoxyuridine (0.2 g) was prepared as follows. 5-cyano-2'-deoxyuridine (0.2 g 0.05 mol) was dissolved in 1 N hydrochloric acid (10 ml). 10% palladium-on-charcoal (0.1 g) was added and the mixture hydrogenated at 2.8 kg/cm 2 for 10 hours at room temperature. The mixture was filtered and the water evaporated under reduced pressure. The residue was triturated with ether, and the product was recrystallized from 80% ethanol.

Example XXX

Maltosetriose was oxidized to it. corresponding carboxylic acid using the following procedure. Maltosetriose (0.5 g, 0.94 mmol) was dissolved in water (5 mL). Lead carbonate (0.42 g, 1.1 mmol) and bromine (0.17 mL, 3.3 mmol) were added,

and the mixture was allowed to react at room temperature for 6 days after which no reducing sugar remained. The mixture was filtered and silver carbonate (0.2 g) added. After new filtration, the filtrate was deionized by elution through "Dowex" 50 (H<+>form). Evaporation of water and drying in the presence of phosphorus pentoxide gave the desired product.

Example XXXI

Maltosetriose was coupled to 5-(3-amino-1-propenyl)-2'-deoxyuridine-5<1>triphosphate by the following procedure. Oxidized maltose triose (190 mg, 0.18 mmol) was dissolved in dimethylformamide (0.8 mL and cooled to 4°C. Isobutyl chloroformate (25 mg, 0.18 mmol) and tri-n-butylamine (43.ul, 0 .38 mmol) was added and the solution was allowed to react at 4 oC for 15 minutes. dissolved in dimethylformamide

(1.2 mL) and 0.1 M sodium borate and cooled to 4°C, was added to the above solution. The mixture was incubated at 4°C for 1 hour and at room temperature for 18 hours. It was then applied to a DEAE-cellulose column and eluted with a gradient of 0.1 to 0.6 M triethylammonium bicarbonate, pH 7.5. The product was finally purified by reverse phase high pressure liquid chromatography.

In the following are Examples XXXII and XXXIII. Example XXXII is a method for labeling allylamine-modified dUTP with a fluorescein substituent. This is an example of the preparation of a self-detecting nucleic acid probe. Example XXXIII is a method for labeling preformed double-helical nucleic acids in the N 2 -position of guanine and in the N -position of adenine. In Example XXXVII, the detector molecule is bound to the probe. Chromosoma 84: 1-18 (1981) and Exp. Cell Res. 128:485-490, describes end-labeling of RNA with rhodamine. However, the method according to the invention is less cleaving and labels internal nucleotides.

Example XXXII

Fluorescein was coupled to 5-(3-amino-1-propyl)-2<1->deoxyuridine-5'-triphosphate (AA-dUTP) as follows. AA-dUTP (10 µmol) dissolved in 2 mL of sodium borate buffer (0.1 M), pH 9.0, was added to fluorescein isothiocyanate (10 mg, 25 µmol) dissolved in 1 mL of dimethylformamide. After 4 hours at room temperature, the mixture was applied to a DEAE cellulose column equilibrated in triethylammonium bicarbonate buffer, pH 7.5. The fluorescein-linked AA-dUTP was purified by elution with a gradient from 0.1 to 0.6 m triethylammonium bicarbonate, pH 7.5.

Example XXXIII

DNA can be modified by reaction with chemical alkylating agents. Lambda DNA was alkylated in the N 2 position in guanine and the N^ position in adenine by reaction of DNA with the aromatic hydrocarbon 7-bromomethylbenz[a]anthracene. 7-Bromomethylbenz[a]-anthracene was obtained as follows. The 7-methyl[a]anthracene in carbon disulfide solution was cooled in a freezing mixture and treated dropwise with one mole equivalent of bromine. After 30 minutes, the product in the suspension was collected, and washed with dry ether and recrystallized from benzene. The yield was 66% with a melting point of 190.5-191.5°C.

DNA, purified from subjects. Lambda, (1.6 mg) was solubilized in

5.0 ml of 20 mM potassium phosphate pH 6.5. To 4.0 ml of the DNA solution was added 500 micrograms of 7-bromomethylbenz[a]-anthracene in dry acetone. After 30 minutes at 20°C, DNA was precipitated with two volumes of cold ethanol. The precipitate was washed sequentially with ethanol, acetone and ether to remove - unbound 7-bromomethylbenz[a]anthracene. Enzymatic hydrolysis of DNA to nucleosides and subsequent chromatography of the products on "Sephadex" LH-20 columns indicated that 18% of the adenine and 48% of the guanine in DNA were modified in

6 2

respectively the N and N -positions.

The modified DNA was made single-stranded either by (1)•heating to 100°C for 5 minutes and rapid cooling or (2) incubation with an equal volume of 0.1M NaOH for 10 minutes and then dialyzing the solution for 4 hours against 1

ml tris-HCl pH 8.0 containing 0.5 ml EDTA to keep the DNA in single-stranded form.

Example XXXIV

A DNA probe was ligated to a synthetic DNA consisting of repeated sequences of E. coli lac operator DNA. After hybridization to detect antiprobe sequences, the hybridized DNA was detected by reaction with biotinylated

lac repressor which in turn was detected by an enzyme-linked immunosorbent assay using goat antibiotin-IGG- to react with the biotin and a second antibody linked to horseradish peroxidase. Lac polyoperator DNA

is described by Caruthers (Second Annual Congress for

Recombinant DNA Research, Los Angeles, 1982), and it was ligated, in a blunt end ligation, using T4 ligase, to an adenovirus DNA probe. In situ hybridization of the polyoperator-labeled probe DNA was performed as described by Gerhard et al ( Proe. Nati. Acad. Sei. USA, 78, 3755 (1981). Biotinylated lac repressor was prepared as described by Manning et al ( Chromosoma , 53 , 107-117

(1075) and was applied to adenovirus infected cells,

fixed to a slide, in Binding buffer consisting of (0.01 Mk Cl, 0.01 M tris (pH 7.6), 0.01 M MgSO , 10~4 MEDTA, 10 MDTT, 5% DMSO (dimethyl sulfoxide) and 50 µg/ml bovine serum albumin from J. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory (1972).The slides were washed in binding buffer to remove unbound biotinylated lac repressor and then probed for biotin using the horseradish peroxidase-linked antibody method. This method can be adapted to create affinity columns where the probe can be bound to immobilized repressor protein and then removed by elution with a specific inducer, e.g., isopropylthiogalactoside or thiomethylgalactoside. The affinity of the repressor operator- the complex is quite

high 10 M. When a specific inducer is bound to'

repressor, the operator-repressor complex collapses. Example XXXV

5-Bromo-2<1->deoxyuridine-5<1->phosphate was prepared as follows: 2<1->deoxyuridine-5'-phosphate (6.2 g) was suspended in a mixture of 60 ml of pyridine and 30 ml of acetic acid. Bromine (0.84 mL) was added with stirring in an ice water bath and stirring was continued for 20 hours at room temperature. The solution was concentrated in vacuo. After redissolving in a minimum of water, a product was precipitated by the addition of ethanol. The crude product was chromatographed on "Dowex" 50 (H ) and eluted with water. The free acid product was precipitated from the concentrated eluent by addition of ethanol.

Example XXXVI -

Alkaline phosphate from calf intestines was biotinylated as follows: The enzyme (1 mg, 7.7 mmol) was chromatographed on

a G-50 column and eluted. with 0.1 M Hepes buffer pH 8.0 containing 0.1 M sodium chloride. The combined fractions were reacted with N-biotinyl-6-amino-caproic acid-N-hydroxysuccinic acid imide ester (0.675 mg, 0.77 μmol) dissolved in 10 ml of dimethylformamide at room temperature for 1 hour. Sodium periodate (0.1 M 125 µl) was added and stirring continued for 2 hours. The mixture was dialyzed at 4 oC overnight in 0.1 M Hepes buffer pH 8.0 with 0.1 M NaCl after which pH

was adjusted to 7.4. Biotin hydrazide (0.1 M, 0.5 ml) dissolved in 0.1 M Hepes buffer pH 7.4 and 0.1 M NaCl was added and the reaction mixture stirred for 30 min at room temperature. The pH was adjusted to 8.0 with 0.2 M sodium carbonate and 0.5 ml of freshly prepared 0.1 M sodium borohydride in water was added, the solution was dialyzed against 0.1 M tris buffer pH 8.0 with 0.1 M NaCl .

Example XXXVII

,6-cyano-2'-deoxyuridine-5'-phosphate was prepared in the same manner as in the method of Veder et al, J. Carbohydr. Nucleosides, Nucleotides, 5, 261 (1978). 5-Bromo-2'-doxyuridine-5'-phosphoric acid (2.0 g, 15 mmol) dried by repeated evaporation from pyridine was dissolved in 50 ml of dimethyl sulphide. Sodium cyanide (490 mg, 10 mmol) was added and the solution stirred at room temperature for 2 days. The solution was diluted with 400 ml of water and the pH adjusted to 7.5. It was applied to a DEAE-cellulose column (HCO 3 form) and washed with 2000 ml of 0.02 M triethylammonium bicarbonate to give the desired product.

\ Example XXXVIII

6-(Methylamino)-21-deoxyuridine-51-phosphoric acid was prepared as follows: 6-cyano2<1->deoxyuridine-5'-phosphoric acid (0.2 g,

60 mmol) was dissolved in 0.1 M hydrochloric acid. After addition

of 10% palladium-on-charcoal (0.1 g) the mixture was hydrogenated

with 2.8 kg/cm 2 in 20 hours at room temperature. The mixture was filtered, neutralized with lithium hydroxide and lyophilized. The product residue was extracted with ethanol.

Example XXXIX

Horseradish peroxidase (20 mg) dissolved in 5 ml of distilled water was added to 1.0 ml of freshly prepared 0.1 M sodium periodate solution. After stirring at room temperature for 20 min. it was dialyzed overnight at 4°C against 1M sodium acetate pH 4.4. Biotin hydrazide (2.6 mg, 5x10-2 mmol) dissolved in

2.0, 0.1 M Hepes buffer pH 7.4 with 0.1 M sodium chloride was brought to pH 8.0 with 0.2 M sodium carbonate and 0.5 ml of a freshly prepared 0.1 M sodium borohydride solution in water was added. After 2 hours at 4°C, the protein was purified

a "Sephadex" G-50 column and eluted with 0.1 M Hepes and 0.1 M NaCl.

Example XL

Carrot acid phosphatase is mentioned by Brunngraber and Chargaff, J. Biol Chem,, (1967) 242, 4834-4840 as a by-product in the purification of phosphortransferase and has been purified to a specific activity of 460 µM/mg/min. at 3 7°C with paranitrophenyl phosphate as substrate. The purification comprised the steps (a) absorption of non-specific proteins by DEA-cellulose, (b) acid purification of the enzyme, (c) acetone fractionation; (d) concanvalin A affinity chromatography, (e) hydroxyapatite chromatography and (f) "Sephadex" G-100 fractionation. The specific activity of the enzyme subjected to the "Sephadex" G-100 fractionation was due to the loss of activity in the previous affinity chromatography step (d) 170 µM/mg/m.- By changing the elution conditions in step (d) , these losses can be avoided with the result that

• the specific activity of the enzyme before the "Sephadex" G-100 fractionation can be improved to 340 uM/mg/m. The "Sephadex" G-100 fractionation step should yield an enzyme with a specific activity of 800 uM/mg/m or higher. Carrot acid phosphatease was biotinylated using the method of

Wilchek et al Biochemistry 6, 247 (1967). To the enzyme

(20 mg) dissolved in 0.1 M NaCl, pH 5, was added biotin hydrazide (2.0 mg, 7x10 -3 mmol) and 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (1 mg , 7x10 mmol) dissolved in 0.1 M NaCl, pH 5. After 2 hours at 4°C, the enzyme was chromatographed on "Sephadex" G-50 and eluted with 0.1 M sodium acetate, pH 5.0.

Of particular importance and significance in the implementation of the present invention is the use of self-signaling or self-indicating or self-detecting. nucleic acids, especially such nucleic acids which can be introduced into double-stranded DNA and the like. Such self-signaling or self-detecting nucleic acids can be produced by covalent binding to an allylamine substituent so that a modified nucleotide according to the present invention is formed, a molecule which will gelate special ions, e.g. heavy metals, rare earths, etc. In general, the gelated ion can be detected either (a) by radioactive emission or (b) by using the ion to catalyze a chromogenic or fluorogenic reaction.

For example, a solution of 3,4-dinitrophenol is reduced to 3,4-diaminocyclohexane

This material is then brominated

to form 3,4-diamino-bromo-cyclohexane (dABCH). This compound is reacted with a halide (Cl, Br, I)-substituted carboxymethyl compound to form a tetra-carboxy-methyl derivative or dABCH (TCM-dABCH): bromine is substituted with an amino group using soluble ammonia

This compound is then reacted with chlorothiophosgene to form the isothiocyanate derivative of (TCM-dANCH). Finally, this compound is reacted with dUTP-allylamine derivative to produce modified dUTP.

Cobalt or other heavy metal ions or other rare earth ions can be gelated to the compound after step 3 above. Or the nucleic acid can be substituted with this adduct and then the ion can be added. (For example, is added

-19

cobalt at pH 6 where the binding constant is 10 M).

Cobalt can be detected by radioactivity. It can too

is demonstrated by its ability to oxidize methylene blue to the leucoform in the presence of molecular oxygen. It can be used to oxidize soluble sulfhydro groups to disulfide bonds, also in the presence of molecular oxygen.

This type of self-signaling molecules can be used

to monitor any nucleic acid hybridization reaction.

It is particularly important for the detection of nucleic acids in

gels (for example in sequencing gels).

As regards its use in radioactivity, it can be used to tailor the desired isotope, i.e. if a strong 3- or y-emitter is needed, this isotope can be gelated. Examples of isotopes that can be used are listed below. Streptavidin, a protein produced by a Streptomyces avidinii is a high molecular weight component of a synergistic pair of compounds that are both present in the culture filtrates of this microorganism. Each of the components in the pair is inactive, but in combination is active against gram-negative microorganisms. It has been found that the small component of this antibiotic prevents the de novo synthesis of the vitamin biotin and thus, at least in synthetic media, shows antimicrobial activity. In complex media, however, the large component must be included to exert the same effect on bacteria. This has been shown to be caused by the presence of external biotin in the complex medium. The large molecular component has been found to bind external biotin and thus shows the same kind of effect as avidin from eggs and oviduct tissue from laying birds.

Streptavidin has been purified and shown to be a 60,000 dalton polypeptide. Like avidin, streptavidin contains four subunits and tightly binds four biotin molecules. Unlike avidin, however, it is non-glycosylated and has a PI of 5.0 compared to avidin which has a PI = 10.5. Because of the difference in pI, streptavidin does not tend to non-specifically react with DNA.

Preparation of streptaydin

A semi-synthetic medium containing salt, 1% glucose,

0.1% asparagine, 0.05% yeast extract and trace elements were prepared. The cultures were grown at 26°C for three days. The mycellium was removed by centrifugation and protein in the supernatant was absorbed onto DEAE cellulose in a batch procedure after the pH was adjusted with IM HCl to 7.2. DEAE cellulose was filtered off and washed with 20 mM Tris-HCl (pH 7.2) until no absorbance could be determined at 280 nm. Streptavidin was eluted with 20 mM Tris-HCl (pH 7.2) containing 0.5 M NaCl. Ammonium sulfate precipitation was used to further concentrate streptavidin (50% w/v at 4°C.

The precipitate was dissolved in water and dialyzed against 1.0 M NaCl,

50 mM Na 2 CO 2 . In the next step, affinity column chromatography on iminobiotin sepharose was used. Eluted streptavidin from the iminobiotin sepharose column was shown to

be chromatographically pure by non-denaturing agarose gel electrophoresis.

The final purification of streptavidin is carried out by affinity purification using an iminobiotin-sepharose column. Iminobiotin is an analogue of biotin in which the carbonyl in the urea part is substituted with an imine function. Iminobiotin will bind avidin and streptavidin at a basic pH, but the complex is dissociable at the acidic pH.

Iminobiotin is produced from biotin in several steps. Biotin is hydrolyzed by barium hydroxide to cis-3,4-diamino-2-tetrahydrothiophene-valeric acid, which is reacted with cyanogen bromide to iminobiotin. The iminobiotin is linked to aminosepharose via the N-hydroxysuccinic acid imide ester of its hydrobromide salt.

The crude protein mixture from DEAE-eluted Streptomyces avidinii incubation media is dissolved in 50 mM sodium carbonate and 1.0 M sodium chloride (pH 11) and applied to an iminobiotin column previously equilibrated with this solution. The column is eluted at pH 11. Streptavidin is then eluted with 50 mM ammonium acetate, pH 4.0 containing 0.5 M sodium chloride. The eluent is dialyzed three times against 1 mM Tris pH 7.4 and lyophilized to dryness.

In the implementation of the present invention, avidin can be used as a detection mechanism for labeled DNA, such as e.g. biotin-labeled DNA. At approximately neutral pH, however, avidin itself forms complexes with DNA, with the result that any signals that could be obtained from a complex between biotin-labeled DNA and avidin may be lost or be undetectable in the background due to the complexation between avidin and unlabeled DNA. This disadvantage of the use of avidin in the execution of the present invention does not occur with streptavidin, which does not form a complex with DNA at approx. neutral pH, but is able to form a complex with the biotin moiety of biotin-labeled DNA.

In another aspect directed at the broad applicability of

avidin and streptavidin for<p>detection of labeled compounds apart from DNA, avidin and streptavidin are particularly effective as a detection mechanism for labeled proteins, polysaccharides and lipids. For example, a special antigen can be attached to a solid matrix and an antibody directed against this antigen, which is itself biotinylated, binds to this antigen.

It can then be examined for the presence of this biotinylated antibody by reacting it with avidin or streptavidin in complex with an enzyme, such as e.g. alkaline phosphatase from calf intestines, or in which a fluorescent molecule is bound, such as e.g. fluoroscein.

The use of the antigen-antibody system for detection of either antigen or antibody is well known. A comparable system is a system based on a glycosylated substrate or molecule and tailored or suitable lectin. In this system, the lectin would carry a label, such as fluorescein or an appropriate enzyme. In this glycosyl-lectin system, the labeled lectin forms a complex with the glycosyl moiety, comparable to the antigen-antibody complex, and this complex comprising the glycosylated molecule and appropriately labeled lectin with the required glycosyl or sugar moiety specificity would then appear by exhibit the desired response from the label portion of the labeled lectin that forms the glycosyl-lectin complex.

Another aspect of the implementation of the present invention which is particularly advantageous is to perform the detection or hybridization in the liquid phase between the DNA

to be detected and the DNA-detecting probe. In this liquid-phase system, neither the DNA molecule to be detected is attached

or the appropriate DNA-detecting probe to some insoluble substrate or some insoluble chemical moiety. The advantages of the liquid phase detection system lie in the speed of the hybridization or hybrid formation between the DNA to be detected and the appropriate DNA probe for this. In a solid-liquid-liquid system, for example, the time required to carry out recognition and hybridization formation is approx. 10 times greater than if it were performed in a completely liquid system, i.e. both the DNA to be detected and the detecting DNA are not attached to an insoluble part.

The probes produced according to the practice of the present invention can be adapted for use in the detection of viruses and bacteria in liquids as indicated above. Where the liquids to be examined do not contain large amounts of protein, viruses therein can be concentrated by absorption on hydroxyapatite and eluted in a small amount of phosphate buffer. When the liquid to be examined contains large amounts of protein, viruses can be concentrated by centrifugation at high speed.

If antibodies were available, the absorption on an affinity column and elution with acid would be preferred, because it would be possible to process many probes according to the embodiment of the invention at the same time. The bacteria to be examined are usually easily concentrated by centrifugation.

According to the implementation of the present invention, identification or characterization of the isolated particles, viruses and bacteria, would be hybridization of the characterizing or identifying DNA thereof with a specific single-stranded DNA probe produced according to the present invention. After hybridization, excess unhybridized probe DNA would be digested with S 2 nuclease and exonuclease I from E. coli at high salt to suppress the cleavage activity of S 2 nuclease, see Vogt, Methods in Enzymology, vol. 65, pages 248-255 (1980). This nuclease treatment would produce mononucleotides from the excess unhybridized, single-stranded DNA probe, but would leave the double-stranded, hybridized DNA intact. This would then be absorbed at high salt content on the "Dowex" anion exchanger (the nucleotides and the small amount of oligonucleotides will not bind to the resin at high salt concentration). The resulting hybridized DNA would then be identified or characterized by various methods applicable to the practice of the present invention.

The special nucleotides according to the invention comprise a phosphoric acid P part, a sugar or monosaccharide S part, a base B part, a purine or a pyrimidine and a signaling chemical part Sig covalently attached thereto, either to P-, S- or The B part. The following are structural formulas for different bases, B-parts and nucleotides that have been modified according to the invention.

The main purines

The special nucleotides according to the present invention, as indicated above, comprise, in addition to the P, S and is parts, a chemical part Sig which is covalently bound to the P, S and/or B parts. Of particular interest consistent with the practice of the present invention will be those nucleotides having the general formula

where P is the phosphoric acid moiety, including mono-, di-, tri-

or the tetraphosphate, S sugar or monosaccharide moiety,

B the base part, either a purine or a pyrimidine. The phosphoric acid part P is attached to the 3' and/or 5<1-> position in the S part when the nucleotide is a deoxyribonucleotide and in the 2',

The 3' and/or 5' position when the nucleotide is a ribonucleotide. The base B part is attached from the N1 position or the N9 position to the 1' position of the S part when the base part

is a pyrimidine or a purine, respectively. The expressed Sig portion is covalently attached to the B portion of the nucleotide and when so attached is capable of self-signaling or making itself self-detecting or its presence known and desirably or preferably allows introduction of the resulting nucleotide P - S - B - Said in or to form a double-stranded helical DNA or RNA or DNA-RNA hybrid and/or to be detectable thereon.

Another special nucleotide according to the invention is characterized by the general formula:

Such nucleotides according to the invention would be characterized as ribonucleotides. The phosphoric acid part is attached in the 2'-, 3'- and/or 5<1-> position in the sugar S part and the base B is attached from the N1 position or the N9 position to the 1' position in the sugar S part when said base is a pyrimidine, respectively

IN

l

or a purine. The chemical Sig moiety is covalently attached to the sugar S moiety and said chemical Sig moiety when attached to said S moiety is able to self-signal or self-detect or make its presence known and preferably allows introduction of the ribonucleotide in its corresponding double-stranded RNA or DNA-RNA hybrid.

Such nucleotides

preferably the chemical Sig-

moiety attached to the C2<1->position in the S moiety or the C3' position in the S moiety.

Nucleotides according to the present invention further comprise those nucleotides which have the formula:

in which P is the phosphoric acid part, S the sugar part and B the base part.

In these special nucleotides, the P part is attached to the 3' and/or 5' position of the S part, when the nucleotide is a deoxyribonucleotide and to the 2', 3' and/or 5' position when the nucleotide is a ribonucleotide . The base B is either a purine or a pyrimidine and the B part is attached from the NI or N9 position to the 1<1-> position in the sugar part when said B part is respectively a pyrimidine or purine. The chemical Sig moiety is covalently attached to the phosphoric acid P moiety via the chemical bond

in said Sig, when attached to said P part ..is capable of self-signaling or making itself manifest or its presence known and desired, the nucleotide is capable

to be introduced into a double-stranded polynucleotide, such as e.g. DNA, RNA or DNA-RNA hybrid and still be self-identifying when introduced as such.

It should be pointed out that the special nucleotides according to the invention which are described or defined above by the general formula P-S-B-Sig also include nucleotides where the chemical Sig part is covalently attached to the B part in the N^ - or 6-amino group position when The B part is adenine or in the N 2 or 2 amino group position when the B part is guanine or in the N 4 or 4 amino group position when the B part is cytosine. The resulting nucleotides containing the Sig portion attached thereto are capable of self-signaling or self-detection or their presence known and detectable is a double-stranded or DNA,

RNA or DNA-RNA hybrid.

As indicated above with regard to the composition of the various special nucleotides according to the invention, the special nucleotides can be summarized as comprising a phosphoric acid part P, a sugar part S and a base part B, a purine or pyrimidine, which combination of P-S-B is well known when it comes to defining nucleotides, both deoxyribonucleotides and ribonucleotides. The nucleotides are then modified according to the invention by having a chemical part Sig covalently bound to the P part and/or the S part and/or the B part. The chemical moiety Sig thus attached to the nucleotide P-S-B can make the resulting nucleotide now comprising P-S-B where the Sig moiety is attached to one or more of the other moieties, self-detecting or self-signaling or capable of its presence known, when introduced into a polynucleotide, especially a double-stranded polynucleotide, such as a double-stranded DNA, a double-stranded RNA or a double-stranded DNA-RNA hybrid. The Sig part should preferably not affect the nucleotide's ability to form a double-stranded polynucleotide containing the mirror Sig-containing nucleotide according to the invention and, when introduced therein, the Sig-containing nucleotide is capable of detection, localization or observation.

The Sig-part used in the production of the special nucleotides according to the invention may comprise an enzyme or an enzyme material, such as e.g. alkaline phosphatase, glucose oxidase, horseradish peroxidase or ribonuclease. The sig part can also>: contain a fluorescent component such as e.g. fluorescein or rhodamine or dansyl. If desired, the Sig part may comprise a magnetic component which is connected to or attached thereto, such as, for example, a magnetic oxide or magnetic iron oxide, which will make the nucleotide or polynucleotide containing such a magnetic part detectable by means of magnetic funds. The Sig part can also include an electron-tight component such as e.g. ferritin,

so that it becomes available for observation. The Sig part can also include a radioactive isotope component such as e.g. radioactive cobalt, making the resulting nucleotide observable by radiation detection agents. The Sig part may comprise a hapten component or in itself be able to form complexes with an antibody specific for this. Most usefully, the Sig part is a polysaccharide or oligosaccharide or monosaccharide, which is able to form complexes with or attach to a sugar- or polysaccharide-binding protein, such as e.g. a lectin, for example Concanavilin A. The Sig component or part of the special nucleotides according to the invention may also comprise a chemiluminescent component.

As indicated according to the embodiment of the present invention

the Sig moiety may comprise any chemical moiety which can be attached either directly or by means of a chemical bond or linker to the nucleotide, such as to the base B moiety thereof, or the sugar S moiety thereof, or the phosphoric acid P moiety thereof.

The Sig component in the nucleotides according to the present invention and the nucleotides and polynucleotides containing the nucleotides according to the invention containing the Sig component are equivalent to and usable for the same purpose as the nucleotides described in the above-mentioned U.S. patent application no. 255,223. More particularly, chemical moiety A is described in U.S. Pat. application no. 255,223 functionally equivalent to the Sig component or chemical part in the special nucleotides according to the invention. The Sig component or chemical part of the nucleotides according to the invention can be covalently attached directly to the P, S or B parts or attached thereto via a chemical bond or binding arm as described in U.S. Pat. patent application 255,223, as indicated by the dotted line connecting B and A in the nucleotides of U.S. Pat. application 255,223. The various linkage arms or linkages identified in the U.S. application no. 255,223

are applicable to and useful in the production of the special nucleotides according to the invention.

A particularly important and applicable aspect of the special nucleotides according to the invention is the use of such nucleotides in the preparation of DNA or RNA probes. Such probes will contain a nucleotide sequence which essentially matches the DNA or RNA sequence in the genetic material to be located and/or identified. The probe will contain one or more of the special nucleotides according to the invention. A probe with a desired nucleotide sequence, such as a single-stranded polynucleotide, either a DNA or RNA probe will then be brought into contact with genetic DNA or RNA material to be identified. Upon localization of the probe and the formation of one double-stranded polynucleotide containing the probe and the appropriate DNA or RNA material to be identified, the resulting double-stranded DNA or RNA-containing material formed will then be observable and identified.

A probe according to the present invention can contain i

substantially any number of nucleotide units, from approx. 5 nucleotides up to approx. 500 or more, like that

might be necessary. It therefore seems that 12 suitable, preferably consecutive, nucleotide units would be sufficient to carry out an identification of most of the DNA or RNA material to be examined or identified, if the sequence of 12 nucleotides in the probe fits a corresponding cooperating sequence in the DNA or RNA material being examined or to be identified. As indicated, such probes can contain one or more of the special Sig-containing nucleotides according to the invention, preferably at least approx.

one particular nucleotide per 5-10 of the nucleotides in the probe.

As indicated above, various techniques can be used in the implementation of the present invention for introducing the special nucleotides according to the invention into DNA and related structures. A particularly useful technique referred to above involves the use of terminal transferase to add biotinylated dUMP to the 3' ends of a polypyrimidine or to single-stranded DNA. The resulting product, such as a single-stranded or cloned DNA, which has biotinylated dUMP attached at the 3' ends,

can be recovered using a Sepharose-avidin column in which avidin will complex with the biotinylated dUMP at the ends of the DNA and then recovered. According to the embodiment of the present invention, hybridization to mRNA can be carried out in solution and the resulting hybrid recovered via a Sepharose-avidin column and the mRNA recovered from there. Similar techniques can be used to isolate the DNA-RNA hybrid.

This technique of using terminal transferase for addition of the special nucleotides according to the invention is widely applicable and the resulting modified nucleotides containing the special nucleotides according to the invention comprising the special biotinylated nucleotides or the special glycosylated nucleotides can be selectively recovered by means of complexation with avidin on a Sepharose-avidin column or complexation with a lectin, such as Concanavalin A or a Sepharose-Concanavalin A column.

As an illustration of the embodiment of the present invention

biotinylated dUTP was added to the 3' ends of dpT^ as well as single- and double-stranded dpT^ using terminal transferase and the resulting product was purified through G-50 "Sepharose" and separated on a Sepharose-avidin affinity column . It was found that 69% of the dpT₂ molecules were biotinylated and recovered on the Sepharose-avidin column. The results of this experiment determined that terminal transferase added biotinylated dUMP to the 3' ends of a polypyrimidine.

Detection of nucleic acids to which particular molecules are covalently attached can be carried out using many naturally occurring proteins to which small molecules are known to bind specifically. In this method, the small molecules are attached to the nucleotide using the allylamine side chain. These nucleotides are then introduced into special nucleic acids using a DNA or RNA polymerase or ligase reaction or a chemical bond. After heat treatment of this probe with a complementary antiprobe sequence, the presence of the probe is determined by the specific binding of the protein to the ligand covalently bound to the probe.

Examples of protein-ligand reactions suitable for this type of detection system include: 1. Enzymes and allosteric effector or modeler molecules. An example of this is the enzyme threonine dehydratase, which is a heterotropic enzyme in that the effector molecule, L-isoleucine, is different from the substrate, L-threonine,

J. Monod, J. Wyman and J.P. Changeux (1965), J. Moi. Biol. 12:88-118.

2. Effector molecules involved in regulation. An example of this is the specific binding of 3',5-cyclic adenosine monophosphate to the cyclic AMP receptor protein, I.Pastan and R. Perlman, Science 169:339-344 (1969). Another example

is the lactose repressor molecule and the inducer molecules isopropylthiogalactoside or thiomethylgalactoside. These two inducer molecules are called grauitous inducer compounds as they are not metabolized by the enzymes they induce, W. Gilbert and B. Muller-Hill, Proe. Nati. Acad. Pollock. (US), 70:3581-3584, (1973). 3. Hormone receptors and other receptors on the surface of the cell to which organic molecules will bind specifically. An example of this is the epinephrine-epinephrine-reactor system in which epinephrine is bound in a stereospecific manner with a high affinity to the receptor. Since the receptor protein is insoluble in water, in this system it will be embedded in a lipid bilayer structure such as e.g. a liposome. Suitable detector systems will include specific enzymes or fluorescent molecules within or within the lipid bilayer. 4. Specific ligand-binding proteins involved in transport of small molecules. An example of this is the periplasmic binding proteins in bacteria which have been shown to bind many amino acids, glucose, galactose, ribose and other sugars, Pardee, A. Science, 162:632-637, (1968), G.L. Hazelbaur and J. Adler, Nature New Bio. 230: 101-104 (1971).

In the examples mentioned above, the ligand that is bound to the nucleic acid reacts with a naturally occurring protein. The special feature of this reaction is due to the ligand binding site in the protein.

A further example of the impact of small molecules

on naturally occurring proteins, it includes the specific binding of coenzymes or other prosthetic molecules to enzymes. Examples of such coenzymes include thiamine pyrophosphate, flavin mononucleotide, flavinadenine dinucleotide, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, coenzyme A, pyridoxyl phosphate, biotin, tetrahydrofol-

acid, coenzyme B, 2, lipoic and ascorbic acid. Many of these molecules form covalent bonds with their apoenzymes. However, some, such as coenzyme A, coenzyme B^°9tetrahydrofolic acid, associate in a non-covalent but specific manner with their cognate apoenzymes. A specific coenzyme-apoenzyme system for use in this system is flavin adenine dinucleotide (FAD) and flavin adenine dinucleotide reductase isolated from Escherichia coli. With this system, the binding of FAD is sufficiently strong to enable detection..

The special nucleotides according to the invention and polynucleotides comprising such nucleotides, either single-stranded or double-stranded polynucleotides, DNA and/or RNA, comprising the components, the phosphoric acid part P, the sugar or monosaccharide part S, the base part B, a purine or a pyrimidine , and the signaling or self-revealing part,

Sig, which is covalently attached to either the P-..S- or B-moieties, as indicated above, has many applications. For example, the nucleotides according to the invention and the polynucleotides containing the nucleotides according to the invention can be used as immune-stimulating agents, as adjuvants in vaccines,

as a means of stimulation or induction from competent cells, such as e.g. lymphocytes, for the production of lymphokines, cytokines or cytokinins, interferon or other cellular products.

It is well known that double-stranded poly A:U is a stimulator or inducer of interferon production, although it is a weak agent. Similarly, poly I:C is also known as a stimulator or inducer for the production of interferon.

The advantage of nucleotides, such as double-stranded polynucleotides containing one or more nucleotides according to the invention, is that such nucleotides will actually be more effective and more powerful inducing or stimulating agents for the production of interferon and related materials from cells. For example, nucleotides according to the invention which contain the above-described components P, S-, B and Sig are suitably prepared so that the nucleotides and polynucleotides prepared therefrom are more resistant to nucleases. Such nucleotides and polynucleotides which contain the same and are suitably prepared are similarly more able to come into contact with, stimulate and penetrate cell surfaces or membranes, such as e.g. the cell surfaces or membranes of lymphocytes and other cells to stimulate them to produce a desired cellular product, such as interferon.

Particularly useful among these special nucleotides according to the invention with the formula P-S-B-Sig and particularly useful are those where the Sig component is in the 5-position of the pyrimidine or the 7-position of the purine or a deazapurine or the N 2-position of guanine or the N-position of adenine. Such nucleotides and polynucleotides which contain the same, both single-stranded and double-stranded nucleotides,

DNA and/or RNA is produced according to the invention to provide increased stability as regards the double-stranded helix in DNA or RNA or DNA-RNA hydride containing the same. Increased resistance to nucleases is also achievable as well as changes or beneficial changes in the hydrophobic properties or the electrical or charge properties of the nucleotides and polynucleotides containing them. Nucleotides and polynucleotides according to the invention are also produced which, when administered to humans, have reduced pyrogenicity or exhibit less of other whole body toxic responses.

In addition, the nucleotides and polynucleotides according to the invention are prepared to provide a ligand, which e.g. the component Sig, to which specific polypeptides can combine to initiate or bring about the formation of RNA complexes. It can therefore be seen that the nucleotides according to the invention comprise P, S, B and Sig constituents in which Sig is covalently bound to either the P, S or B moieties, providing a number of chemical agents such as special biological effects including, therapeutic effects and cytotoxic effects.

The special nucleotides according to the invention, including polynucleotides containing these nucleotides, are, in addition to being stimulating or inducing agents for the production of cellular material or products,

like for example. interferons, lymphokines and/or cytokines,

also applicable because of their chemotherapeutic effect and for the preparation of chemotherapeutic agents based thereon, but also for their cytotoxic effects and the preparation of cytotoxic agents based thereon. The part Sig which is attached to the particular nucleotides of the invention containing the other parts or components P,.S and B provides a site in and of itself for attachment

of special agents with known chemotherapeutic or cytotoxic effects. Such nucleotides can be introduced or administered to the person treated, e.g. human body or an animal, so that they are introduced into the DNA and/or RNA components of the body or cell so that they either affect the synthesis in the body or cellular DNA and/or RNA or to attack tumors or actually kill or otherwise affect on the growth of unwanted cells.

The administration of the nucleotides and/or polynucleotides containing the nucleotides to the body, the human body or the animal, can be carried out in a number of suitable ways. Particularly effective. would the intravenous introduction into the body of the preparation containing the nucleotides according to the invention and a suitable physiologically acceptable carrier be, or the nucleotides could be administered subcutaneously, transdermally or intramuscularly or by direct introduction to the site where the chemotherapeutic or cytotoxic effect of the nucleotides is sought or desired to be efficient. Not only can desired chemotherapeutic or cytotoxic effects be achieved systemically or locally, but also as stated above, the special P-, S-, B- and Sig-containing nucleotides according to the invention, including polynucleotides containing such nucleotides are used

only as immune-stimulating agents and aids therefore. Vaccines containing the special nucleotides and polynucleotides according to the invention can therefore be produced with improved efficiency and versatility.

Of particular interest in the implementation of the present invention, improved polynucleotides containing the special nucleotides according to the invention are used as inducing and stimulating agents for the production of interferon.

Such polynucleotides will be single-stranded or double-stranded ribonucleotides, dsRNA, with the structures,

where A and B are complementary base pairs, such as a purine,

a 7-deazapurine or pyrimidine modified by the addition of an organic part Sig according to the invention in the 5-position of the pyrimidine ring or the 7-position of the purine ring or N 2 in guanine, or N 6 in adenine or N 4 in cytosine as described here. the modifications of the polynucleotides in these positions lead to relatively non-cleavable or non-cleavable double-stranded nucleic acid molecules as measured by association rates and melting points. In the special poly-

the nucleotides according to the invention which are used as inducing agents for interferon and other cellular or humoral factors or components, such as e.g. lymphokines or cytokines, the following groups will be attached to them as indicated by the formulas,

When using the special polynucleotides according to the invention, which e.g. the special dsRNA according to the invention, in the induction process for the production of interferon, it has been shown that DEAE-dextran facilitates this operation. Since DEAE-dextran forms complexes with dsRNA and protects it from nuclease degradation, interferon induction is thereby increased. It is also noted that poly:nC:rI is taken into cells more efficiently when complexed with DEAE-dextran. In the practice of this invention, therefore, the hydrophobic properties and the ionic or electron-charge properties of the particular dsRNA according to the invention are important factors and amenable to manipulation in the utility of these materials to induce interferon production. It has been observed that such conditions or factors which promote the induction of interferon also lead to and promote the induction of other cellular or humoral components, such as e.g. lymphokines and cytokines. It is therefore • clear that the special nucleotides and polynucleotides containing the special nucleotides according to the invention act as immunomodulators and stimulators for other immune responses than just being effective as inducers for interferon production. Good agents for the above according to the implementation of the invention will include nucleotides in which the Sig part contains biotin or streptavidin or avidin.

Poly-rl:poly-rC complexed with poly-L-lysine exhibits auxiliary properties and such properties are increased and improved according to the invention when the poly-rl and poly-rC components are modified so that they comprise one or more of the special nucleotides according to the invention.

The production of DNA probes according to another aspect of the invention can be carried out in a way that does not require the production or use of the special nucleotides described here. For example, double-stranded DNA can be reacted with a carcinogenic or alkylating agent. After the carcinogen has reacted with or alkylated the double-stranded DNA, the resulting modified DNA is melted to prepare a DNA hybridizing probe containing the reaction product between the DNA and the carcinogen or alkylating agent. When thus modified or converted • ■ DNA is used as a hybridizing probe, any resulting double helix or double-stranded DNA formed would be detected or detected by the double-antibody technique. The primary antibody would be an anti-carcinogen and the secondary antibody would be horseradish peroxidase-conjugated anti-peroxidase antibody. The advantage of this technique is that it will be easy to label the double-stranded DNA. This particular method is indicated above in the examples that follow the description of the invention and is generally applicable for the production of DNA probes from double-stranded or double-helical DNA. However, this method is a cleavage method which includes modification of the double-helical deoxyribonucleotide polymer or DNA.

In the description of the special nucleotides and the modified DNA used or developed in the execution of the present invention, mono-, oligo- and polysaccharides are mentioned. It has been pointed out that derivatives of mono-, oligo- and polysaccharides are also usable in the production of the special nucleotides according to the invention. For example, it is possible to modify certain sugar parts that are used in the production of the particular nucleotides and use the resulting modified sugar parts to carry out or carry out additional chemical reactions. Such modified mono-, oligo- and polysaccharide moieties, when used as the Sig-moiety in the preparation of the special nucleotides of the invention, provide increased versatility in the detection of the nucleotides or other compounds containing such modified saccharides, either as the sugar S or as the Sig part thereof.

In another aspect of the invention, the Sig part, instead of being attached to a nucleotide, can also be attached to the protein. Not only can such proteins be attached to nucleotides or polynucleotides, but also such proteins can be identified per se whether they are attached to a nucleotide or polynucleotide or they are not attached. According to the embodiment of this aspect of the invention, a suitable such protein adduct will have the formula

in which R^ is OH or an amino acid or acids and R 2 is the side chain of an amino acid and R^ is H or an amino acid or acids and Sig is attached to R^ and/or R 2 and/or R^•

j

Claims (44)

1. Nucleotide with the general formula P-S-B-Sig, characterized in that Per is the phosphoric acid part, S is the sugar or monosaccharide part and B is the base part, the phosphoric acid part being attached in the 3' and/or 5' position in the sugar part when the nucleotide is a deoxyribonucleotide and in the 2', 3' and/or 5' position when said nucleotide is a ribonucleotide, the base is a purine or a pyrimidine, which is attached from the N1 position or the N9 position to the 1' position in the sugar part when the base is resp. a pyrimidine or a purine and where Sig is a chemical part that is covalently attached to the base B in said nucleotide, Sig when attached to the base B is capable of signaling itself or making itself evident or its presence known. 2. Nucleotide according to claim 1, characterized in that t Sig is attached to B in a position such that the nucleotide is able to be introduced into or to form a double-stranded ribonucleic acid, a double-stranded deoxyribonucleic acid or a double-stranded doxyribonucleic acid-ribonuclein -acid hybrid. 3. Nucleotide according to claim 1, characterized in that Sig is attached to B in a position such that when the nucleotide is introduced into or attached to or connected to a double-stranded deoxyribonucleic acid or double-stranded ribonucleic acid or DNA-RNA hybrid, the aforementioned chemical Share the ability to self-signal or self-identify or make your presence known. 4. Nucleotide according to claim 1, characterized in that Sig is attached to said base B in the C5 position when base B is a pyrimidine or in the C7 position when base B is a deazapurine. 5. Nucleotide according to claim 1, characterized in that the base B is a pyrimidine and the chemical part Sig is attached to the pyrimidine in the N3 position. 6. Nucleotide according to claim 1, characterized in that the chemical part Sig is an aliphatic chemical part containing at least 3 carbon atoms and at least one double bond. 7. Nucleotide according to claim 1, characterized in that the base B is a pyrimidine and the chemical part Sig is attached to the pyrimidine in the C5 position. 8. Nucleotide according to claim 1, characterized in that the chemical part Sig is a sugar selected from the group consisting of triose or tetrose, or pentose, a hexose or heptose, and an octose. 9. Nucleotide according to claim 1, characterized in that the chemical part Sig comprises a component selected from the group consisting of an electron-dense component, a magnetic component, an enzyme, a hormone component, a radioactive component, a metal-containing component, a fluorescent component and an antigen or antibody component. 10. Nucleotide according to claim 1, characterized in that the chemical part Sig is a sugar residue and the sugar is complexed with or attached to a sugar- or polysaccharide-binding protein. 11. Nucleotide according to claim 1, characterized in that the chemical part Sig comprises a radioactive isotope. 12. Nucleotide according to claim 1, characterized in that the chemical part Sig comprises an antigen or hapten component which is capable of forming a complex with an antibody which is specific to said component. 13. Nucleotide according to claim 1, characterized in that the chemical part Sig is connected to the base B by means of a chemical bond. 14. Nucleotide according to claim 1, characterized in that the chemical bond is selected from or comprises a part selected from the group consisting of 15. Polynucleotide linked or attached to a polysaccharide, characterized in that the polynucleotide is terminally ligated to the polysaccharide. 16. Polydeoxyribonucleotide which is linked or attached to a polypeptide, characterized in that the polydeoxyribonucleotide is terminally ligated or attached to said polypeptide. 17. Ribonucleotide with the general formula: characterized by atPer the phosphoric acid part, S the sugar part and B the base part, the phosphoric acid part being attached in 2'-,; the 3'- and/or 5'-position in the sugar moiety, the base B is attached from the Nl-position or the N9-position to the 1 <1-> position in the sugar moiety when the base is a pyrimidine or a purine, respectively, and Sig is a chemical moiety which is covalently attached to the sugar S, as Sig, when attached to the sugar S, is able to self-signal or make self-evident or its presence known. 18. Polyribonucleotide linked or attached to a polypeptide. 19. Polyribonucleotide according to claim 18, characterized in that the polypeptide is terminally attached or ligated to the polyribonucleotide. 20. Nucleotide with the general formula characterized by atPer the phosphoric acid part, The S sugar part and the B base part, the phosphoric acid part being attached to the 3' and/or 5<1> position in the sugar part when the nucleotide is a deoxyribonucleotide and in the 2', 3' and/or 5' position when the nucleotide is a ribonucleotide , the base B is a purine or pyrimidine, the base B being attached from the N1 position or the N9 position to the 1' position in the sugar moiety when the base B is a pyrimidine or a purine, respectively, and Sig is a chemical moiety that is covalently attached to. the phosphoric acid part via the chemical bond as Sig, when it is attached to the phosphoric acid part P, is capable of signaling itself or making itself evident or its presence known. 21. Nucleotide with the general formula characterized in that P is the phosphoric acid moiety, S the sugar and monosaccharide part, and B the base part, the phosphoric acid part being attached to the 3' and/or 5' position in the sugar part when the nucleotide is deoxyribonucleotide and in the 2', 3' and/or 5' position when the nucleotide is a a ribonucleotide, the base is a purine or a pyrimidine, the base being attached from the Nl position or the N9 position to the Cl' position of the sugar moiety when the base is a pyrimidine or a purine, respectively, and Si- is a chemical moiety that is covalent attached to the base B in the nucleotide, Sig being attached to the N - or 6-amino group when the base B is adenine or the N 2 or 2-amino group when the base B is guanine or the N 4- or 4-amino group nrå base B is cytosine, as Sig, when attached to base B, is able to signal itself or make itself evident or its presence known. 22: Nucleotide with the general formula P-S-B, characterized by the atPer phosphoric acid part, the S sugar or monosaccharide part and the B base part, the nucleotide having covalently attached to the P or S or B part a chemical part Sig, and where the the chemical part Sig when attached to the P part is attached to it by the chemical bond, and when Sig is attached to the S part, the S part is a ribose group, the chemical part Sig, when attached to said P, S or B is capable of signaling itself, or making itself self-detecting or its presence known. 23. Polynucleotide comprising one or more nucleotides according to claim 1 or claim 17 or claim 20 or claim 21 or claim 22, characterized in that it is attached or linked to a polypeptide, in that one or more chemical parts Sig are attached to the polypeptide, in that Say, when it is attached to the polypeptide, it is able to signal itself or make itself evident or its presence known. Nucleotide according to claim 1 or claim 17 or claim 20 or claim 21 or claim 22, characterized in that it contains a gelling agent with the following formula where M is H or a substitutable metal. 25. Polynucleotide containing one or more nucleotides according to claim 1 or claim 17 or claim 20 or claim 21 or claim 22, characterized in that the chemical part Sig comprises a gelation agent. 26. The connection characterized in that M is hydrogen or a metal. 27. The connection characterized by atMer hydrogen or a metal. 28. Nucleotide according to claim 1 or claim 17 or claim 20 or claim 21 or claim 22, characterized in that the chemical part Sig comprises the chemical part 29. Amino acid or polypeptide, characterized in that a part of Sig is attached to it. 30. Amino acid or polypeptide according to claim 29, characterized in that said part Sig comprises a saccharide component. 31. Monosaccharide or polysaccharide, characterized in that some Sig is attached to them. 32. Method for detecting a first compound comprising a nucleotide according to claim 1 as part of the first compound, characterized by bringing the first compound into contact with the second compound which is capable of forming a complex with it under suitable conditions, such that the complex is formed, the complex comprising the first compound and the second compound and detecting the complex. 33. Method for determining the presence of a deoxyribonucleic or ribonucleic acid molecule, characterized by forming a double-stranded hybrid polynucleotide duplex comprising a single strand of deoxyribonucleic acid or ribonucleic acid corresponding to or derived from the deoxyribonucleic or ribonucleic acid molecule and a nucleotide according to claim 22 and detect said duplex. 34. Method for diagnosing a genetic disease in a person, characterized by producing a polynucleotide that is complementary to the deoxyribonucleic acid gene sequence of said person that is associated with said genetic disease and includes a compound according to claim 1 introduced therein, bring said polynucleotide under suitable conditions into contact with deoxyribonucleic acid obtained from said person, so that a double-stranded hybrid duplex is formed and detect the presence of the hybrid duplex, the presence or absence of the hybrid duplex indicating the presence or absence of said genetic disease. 35. Method for diagnosing a genetic disease in a person, characterized by producing a polynucleotide that is complementary to the deoxyribonucleic acid gene sequence of said person that is associated with said genetic disease and includes a compound according to claim 20 introduced therein, bring the polynucleotide under suitable conditions in contact with the deoxyribonucleic acid obtained from said person to form a double-stranded hybrid duplex and detect the presence of the hybrid duplex, the presence or absence of the hybrid duplex indicating the presence or absence of said genetic disease. 36. Method for chromosome karyotyping, characterized by producing a series of modified polynucleotides corresponding to a series of defined genetic sequences located on chromosomes, the polynucleotides comprising one or more compounds according to claim 1, bringing the polynucleotides into contact with deoxyribonucleic acid of or obtained from chromosomes to form hybrid duplexes and detect said duplexes whereby the localization of the duplexes on the chromosomes and the localization of the genetic sequences on the chromosomes are thereby determined. 37. Method for identifying or locating hormone receptor sites on the surface of cells, characterized by binding a compound according to claim 1 or claim 17 or claim 20 or claim 21 or claim 22 to the sites under suitable conditions that allow binding of the compound to the receptor sites and detect the compounds bound to the receptor sites. 38. Diagnostic kit which can be used for determining the presence of a nucleic acid-containing organism or the like, characterized in that it comprises a polynucleotide which in its composition contains a compound selected from the compounds in claim 1, claim 17, claim 20, claim 21 or claim 22, and which is complementary to all or an identifiable, distinct or unique part of the nucleic acid contained in said organism and means ■ to detect or express the presence or absence of a resulting hybrid formed between the polynucleotide and the nucleic acid in the organism when the polynucleotide is brought into contact with the nucleic acid in the organism or the like under hybrid-forming conditions. 39. Method for determining or identifying or diagnosing a first compound capable of forming a complex with or binding to a first component in a second compound, the second compound comprising the first component and a second component, and the second component being attached to or complexed with the first component, characterized by contacting the first compound with the second compound, whereby the first component of the second compound forms a complex with the first compound to form a third compound comprising the first compound and the second the compound and contacting the resulting third compound with a fourth compound capable of binding or attaching to the second component of the second compound to form the third compound. 40. Method for determining or identifying or diagnosing in an organism, cellular material or tissue a first compound comprising a receptor molecule with a receptor site that specifically binds to a second molecule, characterized by bringing the organism, material or tissue containing the receptor molecule in contact with a second compound, the second compound comprising a first component capable of forming a complex with or binding to the receptor molecule, the second compound also comprising a second component, which second component is attached to or complexed with the first component, the first component of the second compound forms a complex with the receptor molecule when brought into contact with said receptor molecule to form a third compound comprising the first compound and the second compound, and bringing the third compound into contact with a fourth compound that can bind to or attach s eg to the second component of the second compound to form the third compound, the first component of the second compound being selected from group A consisting of an amino acid, a peptide or a protein, or from group B consisting of a sugar, an oligosaccharide or polysaccharide or from group C consisting of a purine, a pyrimidine, a nucleoside, a nucleotide, an oligonucleotide or polynucleotide, the second component of the second compound being a sugar, an oligosaccharide or a polysaccharide and the fourth compound being selected from the group consisting of a peptide or polypeptide, a lectin or an antibody. 41. Nucleotide according to claim 1, characterized in that the chemical part Sig which is covalently attached to the base B in the nucleotide comprises a coenzyme. 42. Nucleotide according to claim 1, characterized in that the component Sig comprises a radioactive component. 43 Double-stranded polynucleotide, characterized in that it comprises one or more nucleotides according to claim 1 and where the component Sig thereof is radioactive. 44. Polynucleotide containing one or more nucleotides according to claim 1, or claim 17, or claim 20, or claim 21, or claim 22, characterized in that the part Sig is radioactively labeled.